Bifunctional-modified hydrogels

ABSTRACT

Disclosed are hydrogels wherein a polymer matrix is modified to contain a bifunctional poly(alkylene glycol) molecule covalently bonded to the polymer matrix. The hydrogels can be cross-linked using, for example, glutaraldehyde. The hydrogels may also be crosslinked via an interpenetrating network of a photopolymerizable acrylates. The hydrogels may also be modified to have pharmacologically-active agents covalently bonded to the poly(alkylene glycol) molecules or entrained within the hydrogel. Living cells may also be entrained within the hydrogels.

PRIORITY

Priority is hereby claimed to provisional application Ser. No.60/285,782, filed 23 Apr. 2001, the entire contents of which isincorporated herein.

FEDERAL FUNDING

This invention was made with United States government support awarded bythe following agencies: NIH HL63686. The United States has certainrights in this invention.

FIELD OF THE INVENTION

The invention is directed to hydrogels modified using bifunctionalreagents, use of the hydrogels to deliver drugs or otherbiologically-active agents to a mammal in need thereof, compositionscontaining the hydrogels described herein, and devices, such as wounddressings, diapers, catamenial devices, etc., incorporating thehydrogels.

INCORPORATION BY REFERENCE

All of the reference listed in the “References” section are incorporatedherein.

BACKGROUND

Biological systems, such as healing and embryonic development, operateunder spatially- and temporally-controlled orchestration. A myriad ofsignals and cells all act, in space and time, to heal a cut, forexample, or to surround and neutralize a foreign body. The efficacy ofcurrent materials used to construct biomedical devices is limited by alack of multi-functional structures to complement the inherent dynamicsof these biological systems.

For example, most wound dressings provide nothing more than a simplebarrier to shield the wound and to prevent foreign objects from enteringthe would. Other newer types of dressings also include antibiotics toprevent sepsis at the wound site. However, these dressings do notaddress, for example, the exudation which occurs from a wound. Thus,these dressings must be changed often.

Certain biodegradable polymers have been used in burn dressings,hemostatic patches, and the like. These biodegradable polymers provide abarrier and possibly a tissue scaffold for regrowth. However, thesetypes of dressing have no therapeutic effect. While such types ofdressings provide effective barriers to physical disturbance of thewound site, scarring is still extensive.

Despite the extensive investigation of novel wound dressing materials,very few materials are in current clinical use. An ideal functionalwound dressing should have the following properties: It should benon-toxic, biocompatible, and permeable to moisture and gases to absorbwound exudate and toxins as well to maintain appropriate humidity andoxygen levels. It should be porous to prevent swelling of the wound bedand accumulation of the fluid between the wound site and the material.It should be flexible and durable and minimize local inflammation andinfection, thereby promoting new vascularization, re-epithelialization,and normal healing.

Hydrogels are three-dimensional networks capable of absorbing copiousamounts of water. Hydrogels have been explored for many uses, includingdrug delivery devices, wound dressing materials, contact lenses, andcell transplantation matrices. Edible hydrogels, such as gelatin, findextensive use in various food-related applications, such as texturemodification, gelling, clarification of beers and wines, and as medicinecapsules.

SUMMARY OF THE INVENTION

The invention is directed to hydrogels comprising a polymer matrix,preferably gelatin or a synthetic polymer (preferably a biodegradablepolymer, although the polymer may also be non-biodegradable), modifiedto contain bifunctional poly(alkylene glycols) covalently bonded to thepolymer matrix. Heterobifunctional, poly-C₁-C₆-poly(alkylene glycol)molecules, preferably poly(ethylene glycol) molecules (hPEGs), eachhaving an α-terminus and an ω-terminus, are bonded to the polymerbackbone via covalent bonds involving either of the α- or ω-termini. Oneor more biofunctional agents (i.e., pharmacologically-active agents) arethen bonded to the other of the α- or ω-termini (i.e., the free termini)of the hPEGs, thereby yielding a modified, pharmacologically active,homogenous, and covalently-assembled hydrogel. A schematicrepresentation of the preferred embodiment of the invention is shown inFIG. 4.

Any pharmacologically-active agent, without limitation, can beincorporated into the hydrogel, including (by way of illustration andnot limitation) vulnerary agents, hemostatic agents, antibiotics,antithelmintics, anti-fungal agents, hormones, anti-inflammatory agents,proteins, polypeptides, oligonucleotides, cytokines, enzymes, etc.

The hydrogels of the present invention find many uses, the preferred ofwhich is as a functional wound dressing. In this preferred embodiment,the hydrogel contains as a pharmacologically-active agent a vulneraryagent covalently bonded to a biodegradable polymer matrix via adifferentially-modified, α- and ω-substituted PEG linker.

The hydrogels of the present invention may also be incorporated intobandages, surgical and dental wound packing material, diapers andcatamenial devices, and the like.

The novel hydrogel constructs described herein are not physical blends,which are common in the formulation of current biomedical hydrogels;hence, the chemical and physical properties of the subject hydrogels arehomogenous and can be tailored to suit a particular clinical end-pointrequirement. Furthermore, the hydrogel constructs are mechanicallystable because the components are covalently bonded. In addition, thehydrophilicity and flexibility of the porous hydrogel accommodate theabsorption of wound exudate and assist the final removal of the materialfrom the wound site (if necessary or desired). The nature of gelatin andthe porosity of the construct further facilitate the exchange of gasesand allow healing. Most importantly, the presence of hPEG-conjugatedbioactive compounds and the loading of other pharmaceutical compoundswithin the matrix allows for the temporally- and spatially-controlleddelivery of bioactive signals to modulate and complement the dynamics ofthe host healing process.

The present invention offers several key commercial advantages overexisting products. For example, despite the extensive investigation inthe development of novel wound dressing materials, very few materialsare used clinically due to the multiple requirements necessary for afunctional wound dressing. Ideal functional wound dressings must benontoxic, biocompatible, permeable to moisture and gases to absorb woundexudate and toxins, as well as to maintain humidity and oxygen levels.The dressings should be porous to prevent swelling of the wound bed andto prevent accumulation of fluid between the wound site and thematerial. They should be flexible and durable. They should bebiocompatible and minimize local inflammation and infection. They shouldpromote neovascularization, re-epithelialization, and normal healing.The novel multi-functional hydrogels described herein can be made toaddress all of the above requirements for a clinically viable wounddressing material.

Thus in a first embodiment, the invention is directed to a hydrogel thatcomprises a polymer matrix. The preferred polymer matrix containsreactive amino groups. The most preferred polymer matrices are gelatinand collagen. The polymer matrix is modified using a bifunctionalmodifier comprising a poly(alkylene glycol) molecule having asubstituted or unsubstituted α-terminus and a substituted orunsubstituted ω-terminus. At least one of the α- or ω-termini iscovalently bonded to the polymer matrix. The other terminus projectsinto the interior of the hydrogel mass and modifies its physico-chemicalproperties. By controlling the nature of the α- and ω-termini, thephysical and chemical qualities of the resulting hydrogel can bealtered.

Additionally, in the preferred embodiment, the α- and ω-termini aredifferent, and thus are differentially reactive. This enables, forexample, one or more pharmacologically-active agents to be covalentlybonded to one of the α- or ω-termini that is not bonded to the polymermatrix. Alternatively (or simultaneously), one or morepharmacologically-active agents may also be entrained within thehydrogel.

The polymer matrix of the hydrogel may be cross-linked with across-linking reagent such as glutaraldehyde. Cross-linking alters theabsorption characteristics and material strength of the resulting gel.Thus, cross-linking may be desirable where increased mechanical strengthof the gel is required.

As noted above, the α- and/or ω-termini of the hydrogel may besubstituted or unsubstituted. When substituted, it is preferred that thesubstitution is a moiety selected from the group consisting of halo,hydroxy, C₁-C₂₄-alkyl, C₁-C₂₄-alkenyl, C₁-C₂₄-alkynyl, C₁-C₂₄-alkoxy,C₁-C₂₄-heteroalkyl, C₁-C₂₄-heteroalkenyl, C₁-C₂₄-heteroalkynyl,cyano-C₁-C₂₄-alkyl, C₃-C₁₀-cycloalkyl, C₃-C₁₀-cycloalkenyl,C₃-C₁₀-cycloalkynyl, C₃-C₁₀-cycloheteroalkyl, C₃-C₁₀-cycloheteroalkenyl,C₃-C₁₀-cycloheteroalkynyl, acyl, acyl-C₁-C₂₄-alkyl, acyl-C₁-C₂₄-alkenyl,acyl-C₁-C₂₄-alkynyl, carboxy, C₁-C₂₄-alkylcarboxy,C₁-C₂₄-alkenylcarboxy, C₁-C₂₄-alkynylcarboxy, carboxy-C₁-C₂₄-alkyl,carboxy-C₁-C₂₄-alkenyl, carboxy-C₁-C₂₄-alkynyl, aryl, aryl-C₁-C₂₄-alkyl,aryl-C₁-C₂₄-alkenyl, aryl-C₁-C₂₄-alkynyl, heteroaryl,heteroaryl-C₁-C₂₄-alkyl, heteroaryl-C₁-C₂₄-alkenyl,heteroaryl-C₁-C₂₄-alkynyl, sulfonate, arylsulfonate, andheteroarylsulfonate.

Moreover, these moieties themselves may be further substituted. Thus,the moieties on the α-terminus and the ω-terminus when substituted beara substituent selected from the group consisting of alkyl, aryl, acyl,halogen, hydroxy, amino, alkoxy, alkylamino, acylamino, thioamido,acyloxy, aryloxy, aryloxyalkyl, mercapto, thia, aza, oxo, saturatedcyclic hydrocabon, unsaturated cyclic hydrocarbon, heterocycle, aryl,and heteroaryl.

More specifically, the invention is directed to a hydrogel comprising:

a polymer matrix containing reactive amino acid moieties; and

a bifunctional modifier comprising a compound of formula:

wherein at least one of the “A” or “Z” moieties is covalently bonded tothe reactive amino moieties of the polymer matrix; and wherein “A” and“Z” are independently selected from the group consisting of hydrogen,halo, hydroxy, C₁-C₂₄-alkyl, C₁-C₂₄-alkenyl, C₁-C₂₄-alkynyl,C₁-C₂₄-alkoxy, C₁-C₂₄-heteroalkyl, C₁-C₂₄-heteroalkenyl,C₁-C₂₄-heteroalkynyl, cyano-C₁-C₂₄-alkyl, C₃-C₁₀-cycloalkyl,C₃-C₁₀-cycloalkenyl, C₃-C₁₀-cycloalkynyl, C₃-C₁₀-cycloheteroalkyl,C₃-C₁₀-cycloheteroalkenyl, C₃-C₁₀-cycloheteroalkynyl, acyl,acyl-C₁-C₂₄-alkyl, acyl-C₁-C₂₄-alkenyl, acyl-C₁-C₂₄-alkynyl, carboxy,C₁-C₂₄-alkylcarboxy, C₁-C₂₄-alkenylcarboxy, C₁-C₂₄-alkynylcarboxy,carboxy-C₁-C₂₄-alkyl, carboxy-C₁-C₂₄-alkenyl, carboxy-C₁-C₂₄-alkynyl,aryl, aryl-C₁-C₂₄-alkyl, aryl-C₁-C₂₄-alkenyl, aryl-C₁-C₂₄-alkynyl,heteroaryl, heteroaryl-C₁-C₂₄-alkyl, heteroaryl-C₁-C₂₄-alkenyl,heteroaryl-C₁-C₂₄-alkynyl, sulfonate, arylsulfonate, andheteroarylsulfonate; “m” is an integer of from 2 to 8; and “n” is aninteger equal to or greater than 100. In the preferred embodiment, “m”equals 2 and “n” is greater than 2,000.

A second embodiment of the invention is directed to a hydrogel asdescribed above, with the further inclusion of a second polymer matrix.In this embodiment, the second polymer matrix interpenetrates with thefirst polymer matrix. Thus, the first polymer matrix, with its graftedmodifier molecules, interpenetrates and is physically bound within asecond, interpenetrating polymer matrix. In the preferred secondembodiment, the second polymer matrix comprises a photopolymerizedpoly(acrylate), such as an α-acrylate-ω-acrylate-poly(alkylene glycol),trimethylolpropane triacrylate, acrylic acid, and/or acryloyl halide.The second polymer matrix may be a homo-polymer or co-polymer or two ormore monomer types.

As in the first embodiment, the interpenetrating hydrogels may furthercomprise a pharmacologically-active agent covalently bonded to one ofthe α- or ω-termini that is not bonded to the first polymer matrix.

Likewise, all of the hydrogels according to the present invention mayfurther comprise a pharmacologically-active agent or a living cellentrained within the hydrogel.

A third embodiment of the invention is directed to a method of making ahydrogel as described hereinabove. The method comprises reacting apolymer matrix with a bifunctional modifier comprising a poly(alkyleneglycol) molecule having a substituted or unsubstituted α-terminus and asubstituted or unsubstituted ω-terminus, whereby at least one of the α-or ω-termini is covalently bonded to the polymer matrix.

A fourth embodiment of the invention is directed to the method describedin the previous paragraph, and further comprising contacting the firstpolymer matrix with a plurality of monomers and then polymerizing themonomers to yield a second polymer matrix, wherein the second polymermatrix interpenetrates with the first polymer matrix. This embodimentallows for the in situ formation of interpenetrating polymer networks

The hydrogels of the present invention can be used in any applicationwhere hydrogels are currently employed. Thus, the hydrogels of thepresent invention find use as wound dressings, diapers, catamenialdevices, and the like. In one embodiment, the hydrogels are used toadminister a pharmacologically-active agent to a patient in need of thepharmacologically-active agent. In this use, thepharmacologically-active agent either is covalently bonded within thegel or entrained within the gel. The gel is then administered to thepatient, as by packing it into a surgical or traumatic wound.

The hydrogels of the present invention are also useful as scaffolds tosupport living cells. Thus, the hydrogels of the present invention canbe used as biomechanical devices. The hydrogels will support livingcells within the bulk of the gel, thereby providing a three-dimensionalsupport network in which the cells can grow and proliferate. Hydrogelsaccording to the present invention that contain cells can be implantedinto a patient in need of such cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A summary of the chemical reactions and structure of criticalintermediates and final products of M-PEG, CN-PEG, COOH-PEG, or PT-PEG.(1) sodium/naphthalene, THF, room temperature; (2) ethyl bromoacetate,TEA, THF, room temperature; (3) sodium hydroxide solution, reflux; (4)AC, TEA, THF, 10 min room temperature; (5) sodium ethoxide (or sodiummetal), CH₂Cl₂, room temperature; (6) acrylonitrile room temperature;(7) AC, TEA, THF, 10 min room temperature; (8) TEA, thionyl bromide,toluene, reflux; (9) p-toluenesulfonylchloride, TEA, CH₂Cl₂, roomtemperature; (10) AC, TEA, THF, 10 min room temperature; (11) potassiumphthalimide, CH₂Cl₂, reflux; (12) AC, TEA, THF, 10 min room temperature

FIGS. 2A and 2B. HPLC chromatogram of (2A) evaporative light scatteringdetector signals and (2B) UV signals at 254 nm for PEGdiols and variousXPEGmA. Samples were analyzed with a reverse phase HPLC system (10% to100% acetonitrile at a flow rate of 1 ml/min in 30 min with Jordi 500 Acolumn on a Gilson system) coupled to UV/Vis (200 nm and 254 nm),photodiode array, and evaporative light scattering detectors.

FIGS. 3A and 3B. Surface hydrophilicity of the XPEGmA-co-Ac-co-TMPTAnetwork containing XPEGmA of various concentration, terminal moiety, andmolecular weight. (3A) 2 KDa XPEGmA and (3B) 5 KDa XPEGmA. Legend:♦=M-PEG; ▪=CN-PEG; ▴=COOH-PEG; and ●=PT-PEG.

FIG. 4. A schematic representation of hydrogels according to the presentinvention.

FIG. 5. Graph depicting representative swelling/degradation kinetics.Time in hours is shown on the X-axis; swelling ratio is shown on theY-axis. Key: G, 0.01% glutaraldehyde cross-linked=♦; 10% PG, 0.01%glutaraldehyde cross-linked=▪; 40% EG, 0.01% glutaraldehydecross-linked=▴.

FIG. 6. A schematic representation of an interpenetrating networkhydrogel according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Poly(alkylene glycols), such as poly(ethylene glycol) (PEG), areemployed extensively in a number of medical and pharmaceutical fieldsdue to their low toxicity, good biocompatibility, and excellentsolubility⁽¹⁻⁵⁾. For sake of expository brevity, the followingdescription shall be limited to gels modified by bifunctionalpoly(ethylene glycol) molecules. The invention, however, will functionwith equal success using any poly(alkylene glycol).

Thus, it is preferred that the bifunctional modifier comprise apoly(alkylene glycol) of the formula:

where “m” is an integer of from 2 to 8; “n” is an integer equal to orgreater than 100; and “A” and “Z” are as described above. In thepreferred embodiment, “m” equals 2 and “n” is sufficiently large toyield a PEG molecule having a molecular weight of roughly 100,000 Da.Thus, it is preferred that “n” is greater than 2,000. The “n”substituent may also be sufficiently large to yield a PEG moleculehaving a molecular weight greater than 1×10⁶ Da, in which case “n” isgreater than roughly 20,000.

While having good biocompatibility and solubility, the hydroxyl groupsin PEG-diols or monomethoxy-PEGs have very limited chemical activity.The present invention thus is drawn to novel hydrogels that usebifunctional PEGs and hetero-bifunctional PEGs (“hPEGs”) as covalentgrafts to modify the physical and biological properties of hydrogels.These bifunctional PEGS having improved reactivity and physicochemicalproperties can thus be used to modify polymer matrices in general, andproteinaceous matrices in particular, to yield novel hydrogels. Thesenovel hydrogels are useful in wide array of biomaterial andbiopharmaceutical compositions and devices that include a hydrogelcomponent, including time-release vehicles, wound dressings and packing,bandages, burn dressings, catamenial devices, diapers, etc.

Currently, the synthesis of hPEGs is classified into two generalcategories: 1) statistical terminal modification of PEG precursors; and2) ethylene oxide polymerization methods using special initiators.⁽⁶⁻⁹⁾Although various hPEGs are currently available commercially (e.g., fromShearwater Corporation, Huntsville, Ala.), their high cost and limitedquantity greatly restricts the extensive utilization of such materialsby laboratories in developing novel biomaterials for variousapplications. In developing the present invention, a number of syntheticschemes were developed to produce a library of hPEG compounds based onthe statistical terminal modification method.

A distinct benefit of the various reaction schemes is that they use as astarting material commercially-available PEG-diols. PEG-diol isavailable in a host of different molecular weights, and from a largenumber of international suppliers (including Shearwater Corporation).

Moreover, the synthetic strategy is streamlined so modifications tovarious intermediates results in the formulation of different hPEGproducts.

Using the hPEGs of the present invention, polymer networks havingdiverse physicochemical and surface properties were developed. Thesenetworks can be used to study cell-material interaction.⁽¹⁰⁻¹³⁾

In the Examples that follow, hPEGs were utilized to modify a polymermatrix to yield novel hydrogels. The effect of hPEG concentration,molecular weight, and terminal chemical functionality on the surfacehydrophobicity and cell interaction with the hydrogels was investigatedand is presented in the Examples.

Multiple heterogeneous PEG modifications (e.g., carboxylic acids of thepoly-acrylic acid backbone and the functional group at the danglingterminus of hPEG grafted at the pendent chain configuration) can beemployed to bind several distinct types of biofunctional molecules suchas peptides and pharmaceutics to the hydrogel.

These components therefore are highly useful as functional wounddressings. In the preferred embodiment, the polymer matrix is a modifiedgelatin. The use of gelatin is not incidental. Gelatin is awell-characterized, FDA approved, biodegradable biomaterial. Thus,hydrogels made from modified gelatin are likely to pass regulatorymuster due to the known safety of gelatin.

The hydrophilicity and porosity of gelatin was modified using ampholyticmoieties such as ethylenediaminetetracetic dianhydride (EDTAD). Theresulting polymer backbone can be cross-linked with small amounts ofglutaraldehyde and subsequently loaded with pharmaceutical agents suchas antibiotic drugs. The water-uptake, swelling, degradation, and drugrelease kinetics of the resulting hydrogel can be controlled by varyingthe amount of cross-linking and the extent of EDTAD modification.

To improve its biocompatibility and mechanical properties, the hydrogelwas then grafted with various hPEGs, as described hereinbelow.

To investigate the functional properties of these novel biomaterials,the interaction of hPEGs, hPEG-modified gelatin hydrogels, and syntheticpolymer networks containing human white blood cells and fibroblasts wereexamined, both in vitro and in vivo. The terminal group of the hPEGs hasalso been used to link bioactive peptides to the hydrogel matrix,thereby to control the interaction of host cells such as white bloodcells and to enhance favorable biological interactions. It has beendemonstrated in the Examples that the molecular interaction of severalbioactive oligopeptides in modulating white blood cell behavior and hostinteraction in vitro and in vivo can thus be modified.

The resulting hydrogels can be used a functional wound dressings,bandages, and the like. These functional wound dressings are suitablefor use both internally and externally. The gelatin-hPEG hydrogels ofthe present invention have been tested in a subcutaneous caged implantmodel.

One notable aspect of the hydrogels of the present invention is that thepolymer constructs are not physical blends. The present hydrogels arechemically and physically homogenous and can be tailored to suit aparticular clinical endpoint requirement. The hydrogel is mechanicallystable because the components are covalently bonded together.Additionally, the hydrophilicity and flexibility of the porous hydrogelaccommodates the absorption of wound exudate, blood, etc., and assistsin the final removal of material from the wound site.

The nature of gelatin and the porosity of the construct also facilitatesthe exchange of gases and promotes rapid healing. Most importantly, thepresence of hPEG-conjugated bioactive compounds within the hydrogelmatrix itself adds qualitative value and control to the wound healingprocess.

As described hereinbelow, a synthetic scheme was developed to created alibrary of heterobifunctional PEGs (hPEGs) having two distinct terminalmoieties. The hPEGs were then used to make modified polymer hydrogelshaving various surface and physicochemical properties. Extensive NMR andHPLC analyses confirmed the chemical structure of hPEG. Thehydrophilicity of the polymer network was predominantly dependent on thehPEG concentration, with the molecular weight of the starting,unmodified PEG and the terminal functional groups also playing roles.Adherent human fibroblast density on the hydrogels remained constantwith increasing hPEG concentration in the gel formulation but decreasedrapidly on hydrogels containing 0.8 to 1.25 g/ml of hPEGs. This trendwas independent of the hPEG terminal moiety and molecular weight. Noadherent cells were observed on all sample gels containing 2.5 g/ml ormore of hPEGs.

Abbreviations and Definitions:

“Ac”=acrylic acid

“AC”=acryloyl chloride (CAS No. 814-68-6)

“CHD”=chlorhexidine digluconate

“CN-PEG”=α-cyanoethyl-ω-acrylate-PEG

“COOH-PEG”=α-carboxyl-ω-acrylate-PEG

“EDTAD”=ethylene diaminetetracetic dianhydride

“hPEG”=heterobifunctional PEG

“IPN”=interpenetrating network hydrogels

“mPmA”=α-methyl-ω-aldehyde-PEG

“mPEG”=α-methoxy-PEG

“M-PEG”=α-methyl-ω-acrylate-PEG

“PEG” and “PEG diol”=polyethylene glycol

“PEGdA”=PEG-diacrylate

“PEG dial”=α-aldehyde-ω-aldehyde-PEG

“PT-PEG”=α-phthalimide-ω-acrylate-PEG

“TEA”=triethylamine

“THF”=tetrahydrofuran

“TMPTA”=trimethylolpropane triacrylate (i.e.,2-ethyl-2-(hydroxymethyl)-1,3-propanediol triacrylate, CAS No.15625-89-5)

“XPEGmA”=hPEG with acrylate ω-terminal and α-terminal of differentmoiety

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a fully saturated, straight, branched chain, orcyclic hydrocarbon radical, or combination thereof, and can include di-and multi-valent radicals, having the number of carbon atoms designated(e.g., C₁-C₁₀ means from one to ten carbon atoms, inclusive). Examplesof alkyl groups include, without limitation, methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)ethyl, cyclopropylmethyl, and homologs and isomers thereof,for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Theterm “alkyl,” unless otherwise noted, also includes those derivatives ofalkyl defined in more detail below as “heteroalkyl” and “cycloalkyl.”

The term “alkenyl” means an alkyl group as defined above containing oneor more double bonds. Examples of alkenyl groups include vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), etc., and the higher homologs and isomers.

The term “alkynyl” means an alkyl or alkenyl group as defined abovecontaining one or more triple bonds. Examples of alkynyl groups includeethynyl, 1- and 3-propynyl, 3-butynyl, and the like, including thehigher homologs and isomers.

The terms “alkylene,” “alkenylene,” and “alkynylene,” alone or as partof another substituent means a divalent radical derived from an alkyl,alkenyl, or alkynyl group, respectively, as exemplified by—CH₂CH₂CH₂CH₂—.

Typically, alkyl, alkenyl, alkynyl, alkylene, alkenylene, and alkynylenegroups will have from 1 to 24 carbon atoms. Those groups having 10 orfewer carbon atoms are preferred in the present invention. The term“lower” when applied to any of these groups, as in “lower alkyl” or“lower alkylene,” designates a group having eight or fewer carbon atoms.

“Substituted” refers to a chemical group as described herein thatfurther includes one or more substituents, such as lower alkyl, aryl,acyl, halogen (e.g., alkylhalo such as CF₃), hydroxy, amino, alkoxy,alkylamino, acylamino, thioamido, acyloxy, aryloxy, aryloxyalkyl,mercapto, thia, aza, oxo, both saturated and unsaturated cyclichydrocarbons, heterocycles and the like. These groups may be attached toany carbon or substituent of the alkyl, alkenyl, alkynyl, alkylene,alkenylene, and alkynylene moieties. Additionally, these groups may bependent from, or integral to, the carbon chain itself.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable, saturated or unsaturated,straight, branched chain, or cyclic hydrocarbon radical, or combinationsthereof, consisting of the stated number of carbon atoms and from one tothree heteroatoms selected from the group consisting of O, N, Si, and S,and wherein the nitrogen and sulfur atoms may optionally be oxidized andthe nitrogen heteroatom(s) may optionally be quaternized. Theheteroatom(s) O, N and S may be placed at any interior position of theheteroalkyl group. The heteroatom Si may be placed at any position ofthe heteroalkyl group, including the position at which the alkyl groupis attached to the remainder of the molecule. Examples include—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂—S (O)—CH₃, —CH₂—CH₂—S (O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may beconsecutive, such as in —CH₂—NH—O—CH₃ and —CH₂—O—Si(CH₂)₃. Explicitlyincluded within the term “heteroalkyl” are those radicals that couldalso be described as “heteroalkylene” (i.e., a divalent radical, seenext paragraph), and “heterocycloalkyl” (i.e., containing a cyclicgroup). The term “heteroalkyl” also explicitly includes unsaturatedgroups (i.e., heteroalkenyls and heteroalkynyls).

The term “heteroalkylene” by itself or as part of another substituentmeans a divalent radical derived from heteroalkyl, as exemplified by—CH₂—CH₂—S—CH₂CH₂—and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups,heteroatoms can also occupy either or both of the chain termini. Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied.

The term “aryl” is used herein to refer to an aromatic substituent,which may be a single aromatic ring or multiple aromatic rings which arefused together, linked covalently, or linked to a common group such as adiazo, methylene or ethylene moiety. The common linking group may alsobe a carbonyl as in benzophenone. The aromatic ring(s) may include, forexample phenyl, naphthyl, biphenyl, diphenylmethyl and benzophenone,among others. The term “aryl” encompasses “arylalkyl” and “substitutedaryl.” For phenyl groups, the aryl ring may be mono-, di-, tri-, tetra-,or penta-substituted. Larger rings may be unsubstituted or bear one ormore substituents.

“Substituted aryl” refers to aryl as just described including one ormore functional groups such as lower alkyl, acyl, halogen, alkylhalo(e.g., CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy,phenoxy, mercapto, and both saturated and unsaturated cyclichydrocarbons which are fused to the aromatic ring(s), linked covalentlyor linked to a common group such as a diazo, methylene, or ethylenemoiety. The linking group may also be a carbonyl such as in cyclohexylphenyl ketone. The term “substituted aryl” encompasses “substitutedarylalkyl.”

The term “acyl” is used to describe a ketone substituent, —C(O)R, whereR is substituted or unsubstituted alkyl, alkenyl, alkynyl, or aryl asdefined herein.

The term “carbonyl” is used to describe an aldehyde substituent. Theterm “carboxy” refers to an ester substituent or carboxylic acid, i.e.,—C(O)O— or —C(O)—OH.

The term “halogen” or “halo” is used herein to refer to fluorine,bromine, chlorine and iodine atoms.

The term “hydroxy” is used herein to refer to the group —OH.

The term “amino” is used to designate NRR′, wherein R and R′ areindependently H, alkyl, alkenyl, alkynyl, aryl or substituted analogsthereof. “Amino” encompasses “alkylamino,” denoting secondary andtertiary amines, and “acylamino” describing the group RC(O)NR′.

The term “alkoxy” is used herein to refer to the —OR group, where R isalkyl, alkenyl, or alkynyl, or a substituted analog thereof. Suitablealkoxy radicals include, for example, methoxy, ethoxy, t-butoxy, etc.The term “alkoxyalkyl” refers to ether substituents, monovalent ordivalent, e.g. —CH₂—O—CH₃ and —CH₂—O—CH₂—.

The term “gelatin” as used herein means any and all kinds of gelatin, ofany type (e.g., Type A from pork, with an isoelectric point betweenabout 7.0 and 9.0, and Type B from beef with an isoelectric point ofapproximately 5.0), from any source, of any bloom value, acid- oralkaline-treated, etc., without limitation. The “bloom strength” of agelatin is defined as the force required for a plunger of defined shapeand size to make a 4 mm depression in a gel that has been prepared at6.67% w/w concentration and chilled at 10° C. in a bloom jar for 16-18hours. The force is recorded in grams. Commercially, gelatin isavailable from a host of commercial suppliers. At commodity amounts andprices, gelatin is generally available with bloom strengths ranging fromabout 50-300 bloom. Such gelatins are available from, for example,Leiner Davis Gelatin, a wholly-owned subsidiary of Goodman FielderIngredients of Sydney, Australia. Gelatins having bloom values outsidethis range are also available as specialty chemicals and are includedwithin the scope of the term “gelatin.” For example a zero bloom(non-gelling) gelatin is available from Great Lakes Gelatin Co.,Grayslake, Ill.

Likewise, the term “collagen” as used herein means any and all kinds ofcollagen, of any type, from any source, without limitation. Cross-linkedcollagen, esterified collagen, and chemically-modified collagen, such asthat taught by U.S. Pat. No. 4,390,519, are included with the term“collagen.”

The term “polymer matrix” encompasses any type of polymer matrix thatcan function as a hydrogel, including, without limitation, gelatin,calcium alginate, calcium/sodium alginate, collagen, oxidizedregenerated cellulose, carboxymethylcellulose, amino-modifiedcelluloses, such as6-deoxy-6-(4-aminophenyl)-amino-2(3)-O-tosylcellulose, whey proteingels, and the like.

The term “photopolymerizable acrylate” refers to any acrylate-containingmolecule capable of being photopolymerized, without limitation.Expressly included within this definition are bis-diacrylate-PEGs, i.e.,poly(alkylene glycol) molecules having an α-acrylate moiety and anω-acrylate moiety. TMPTA is also a photopolymerizable acrylate.

Modified PEGs:

Commercial PEG-diols can be purchased essentially as a commodity item,in large amounts and at relatively inexpensive prices. The first step inmodifying the α- and ω-termini of the PEG-diols is to convert them inaldehyde groups. This is very easily accomplished by treating thePEG-diol with acetic anhydride:

The reaction is very facile and quantitative.

With the PEG-dialdehyde in hand, the molecule can be further modifiedusing any of the routes shown in FIG. 1, among many others. For example,as shown in FIG. 1, the PEG-diol can be converted into anα-hydroxy-ω-carboxy-PEG, which can then be converted into anα-acrylate-ω-carboxy-PEG. Or the PEG-diol can be converted into aα-hydroxy-ω-cyanoethyl-PEG, which can then, in turn, be converted into aα-acrylate-ω-cyanoethyl-PEG.

The PEG-diol can be directly converted, by simple halogenation of thehydroxy group to α-hydroxy-ω-halo-PEG. The PEG diol can also betosylated and acrylated to thereby yield α-acrylate-ω-tosylated-PEG. Thetosyl group can be exchanged for a succinimidyl or phthalimidyl or othernitrogen-containing heterocycle group. α-Hydroxy-ω-methoxy-PEG can beconverted directly into α-acrylate-ω-methoxy-PEG. See FIG. 1. (See alsoHern & Hubbell, (1998) J. Biomed. Mater. Res. 39:266-276; Morpurgo etal. (1996) App. Biochem. Biotech. 56:59-72; and Abuchowski et al. (1984)Cancer Biochem. Biophys. 7:175-186.)

Thus, for example, α-hydroxy-ω-glutarate-PEG can be synthesized bytreating a PEG-diol with glutaric anhydride and glutaric acid in THFwith gentle heating:

The glutaric anhydride and the glutaric acid are added and the solutiongently heated to 55° C. The solution is maintained at that temperature,with stirring, for one day. The solution is then cooled to roomtemperature and filtered. The filtrate is then precipitated in coldhexane, the resulting precipitate is then removed by filtration, anddried in a vacuum to yield the desired product, generally a mixture ofPEG-bis-glutarate and α-hydroxy-ω-glutarate-PEG. The two can beseparated chromatographically (see the Examples).

The glutarate group can be further reacted to add a nitrogen-containingheterocycle, such as a succinimidyl group by reacting the.α-hydroxy-6-glutarate-PEG with N-hydroxy-succinimide in the presence ofa water-soluble carbodiimide:

The N-hydroxy-succinimide is added and the solution cooled to 0° C. Thedicyclohexylcarbodiimide (DCC) is added dropwise and the solutionstirred for one day and filtered. The filtrate is precipitated by addingcold hexane. The resulting precipitate is filtered and dried in avacuum. This yields the desired product, generally a mixture ofPEG-bis-N-succinimidylglutarate andα-glutarate-ω-succinimidylglutarate-PEG (orα-hydroxy-ω-succinimidylglutarate-PEG, depending upon the startingmaterial chosen). The two can be separated chromatographically (see theExamples).

The α-hydroxy-ω-succinimidylglutarate can be further reacted to yieldα-acrylate-ω-succinimidylglutarates by reacting theα-hydroxy-ω-succinimidylglutarate with acrylic acid in the presence ofTEA.

The PEG molecules may also be modified to introduce other amide bondsinto the molecule. The formation of an amide bond is, of course,extremely useful in modifying the PEG molecule to contain an amino acid,peptide, or protein terminus. Thus, for exampleα-succinimidylglutarate-ω-tryptophanylglutarate PEG can be synthesizedby dissolving the peptide or amino acid in 0.1 M2-(N-morpholino)-ethanesulfonic acid (MES) at 0° C.α,ω-Bis-N-succinimidylglutarate-PEG is added dropwise to the solutionwith constant stirring. The reaction is allowed to continue at 0° C. for1 hour and then allowed to come to room temperature with constantstirring for 4 hours. The reaction solution is then dialyzed against 50volumes of deionized water and the resulting solution lyophilized. Thisyields the desired α-N-succinimidylglutarate-ω-tryptophanylglutarate inrougly 40% yield.

The modified PEGs can be attached to a polymer matrix containingamino-reactive groups using the same procedure as in the previousparagraph, thereby grafting the modified PEG to the amino-reactivegroups of the polymer matrix. See also the Examples. In short, the mono-or dialdehyde-PEG is first dissolved in water. A separate aqueoussolution of NaCNBH₃ is also prepared. The two solutions are then addedsimultaneously to a dilute (5%) solution of gelatin in water. Thereaction is allowed to proceed overnight with gentle heating (50 to 60°C.). The modified gelatin is then separated by filtration.

Using these various synthetic schemes, the following modified PEGmolecules have been made and used to modify gelatin to yield novelhydrogels that fall within the scope of the present invention:Series 1: Alpha-Methoxy Heterobifunctional PEG Derivatives:

Series 1 Chemistry:

1.1. α-Methoxy, ω-hydroxy-PEG is commercially available (Shearwater).

1.2. To synthesize α-methoxy, (ω-tosyl PEG, PEG (1 eq.) was dissolved indry methylene chloride MC followed by addition ofp-toluenesulfonylchloride (1 eq.) and triethylamine (1 eq.). Thesolution was stirred at room temperature for 48 hr, precipitated in coldhexane, collected by filtration and dried in vacuum oven for 24 hr.

1.3. To synthesize α-methoxy, ω-phthalimidyl PEG, α-methoxy, ω-tosyl PEG(1 eq.) from series 1-2 and potassium phthalimide (1.2 eq.) weredissolved in toluene and stirred at 50° C. for 20 hr. The solution wascooled down, filtered and the filtrate was precipitated in cold hexane,collected by filtration and dried in vacuum oven for 24 hr.

1.4. To synthesize α-methoxy, ω-cyanoalkyl PEGs, PEG (1 eq.) wasdissolved in dry MC solution followed by the addition of fine sodiummetal (1.5 eq.) and stirred for 12 hr at room temperature. Excess amountof acrylonitrile was added to the solution, stirred for 12 hr, filtered,and dried by rotary evaporation.

1.5. To synthesize α-methoxy, ω-carboxy PEG, PEG (1 mol) was dissolvedin dry THF. Sodium (1.2 eq.) and naphthalene (1.2 eq.) were dissolved indry THF and stirred under argon for 1 hr. The sodium/naphthalenesolution was added dropwise to the PEG solution and the solution wasstirred under argon for 4 hr.

Ethyl bromoacetate (1.2 eq.) was then added and the solution was stirredunder argon for 12 hr. The solution was filtered and the filtrate wasprecipitated in cold hexane, collected by filtration and dried in vacuumoven for 24 hr. The dried substance was dissolved in deionized waterfollowed by addition of sodium hydroxide (1 eq.). The solution wasstirred at 40° C. for two hr, extracted by MC or two times andevaporated by rotary evaporation.

1.6. To synthesize α-methoxy, ω-glutarate PEG, PEG (1 eq.) was dissolvedin dry THF followed by addition of glutaric anhydride (1.5 eq.) andglutaric acid (0.001 eq.). The solution was stirred at 70° C. for 48 hr,cooled down, precipitated in cold hexane, collected by filtration anddried in vacuum oven for 24 hr.

1.7. To synthesize α-methoxy, ω-triethoxysilane PEG, α-methoxy,ω-acrylate PEG (1 eq.) from series 2-8 was dissolved in dry THF followedby addition of triehyoxysilane (5 eq.) and chloroplatinic acid (agrain). The solution was stirred at 60° C. for 48 hr, cooled down,precipitated in cold hexane, collected by filtration and dried in vacuumoven for 24 hr.

1.8. To synthesize α-methoxy, ω-acrylate PEG, PEG (1 eq.) was dissolvedin dry THF followed by the addition of acryloyl chloride (2 eq.) andtriethylamine (2.2 eq.). The solution was stirred at room temperaturefor 2 hr, filtered and the filtrate was precipitated in cold hexane,collected by filtration and dried in vacuum oven for 24 hr.

1.9. To synthesize α-methoxy, ω-aldehyde PEG, PEG (1 eq.) was dissolvedin DMSO and the solution was added dropwise to the acetic anhydride (20eq.) and stirred at room temperature for 2 hr. Ether was then added tothe solution and stirred for 5 min at room temperature and placed in the−20° C. freezer for 5 minutes to precipitate. The precipitate wascollected by filtration and then dissolved in minimal amounts ofmethylene chloride and reprecipitated likewise twice by ether. Theprecipitate was dried in vacuum oven for 24 hr.

1.10. To synthesize α-methoxy, ω-halo PEG, PEG (1 eq.) was dissolved intoluene followed by addition of triethylamine (1.2 eq.). The solutionwas stirred at 60° C. for 30 min followed by addition of thionyl bromide(1.2 eq.) and stirred at 60° C. for 1 hr. The hot solution was filteredthrough celite and the filtrate was kept in refrigerator at −4° C. for24 hr. The precipitate was collected by filtration and dried in vacuumoven for 24 hr.

1.11. To synthesize α-methoxy, ω-succinimidylglutarate-PEG, α-methoxy,ω-glutarate PEG (1 eq.) from series 1.6 and dicyclohexylcarbodiimide(DCC 1.2 eq.) was dissolved in dry THF respectively. N-hydroxysuccinimide (1.2 eq.) was added to the PEG solution followed by dropwiseaddition of DCC solution. The mixture solution was stirred at roomtemperature for 6 hr, filtered and the filtrate was precipitated in coldhexane, collected by filtration and dried in a vacuum oven for 3 daysand then stored under argon at −4° C. in the refrigerator.

1.12. To synthesize α-methoxy, ω-succinimidylglutrate PEG, α-methoxy,ω-succinimidylglutrate PEG (1 eq.) from series 1.11 was dissolved in DMFfollowed by addition of tryptophan (1.5 eq.). The solution was stirredunder argon for 24 hrs, dialyzed in deionized water and dried bylyophilizer for 3 days.Series 2: Alpha-Hydroxy Heterobifunctional PEG Derivatives:

Series 2 Chemistry:

2.1. PEG is commercially available.

2.2. To synthesize α-hydroxy, ω-tosyl PEG, PEG (1 eq.) was dissolved indry methylene chloride followed by addition of p-toluenesulfonylchloride(1 eq.) and triethylamine (1 eq.). The solution was stirred at roomtemperature for 48 hr, precipitated in cold hexane, collected byfiltration and dried in vacuum oven for 24 hr.

2.3. To synthesize α-hydroxy, ω-phthalimidyl PEG, α-hydroxy, ω-tosyl PEG(1 eq.) from series 2.2 and potassium phthalimide (1.2 eq.) weredissolved in toluene and stirred at 50° C. for 20 hr. The solution wascooled down, filtered and the filtrate was precipitated in cold hexane,collected by filtration and dried in a vacuum oven for 24 hr.

2.4. To synthesize α-hydroxy, (o-cyanoalkyl PEGs, PEG (1 eq.) wasdissolved in dry methylene chloride solution followed by the addition offine sodium metal (1.2 eq.) and stirred for 12 hr at room temperature.Excess amount of acrylonitrile was added to the solution, stirred for 12hr, filtered, and dried by rotary evaporation.

2.5. To synthesize α-hydroxy, ω-carboxy PEG, PEG (1 mol) was dissolvedin dry THF, sodium (1.2 eq.) and naphthalene (1.2 eq.) were dissolved indry THF and stirred under argon for 1 hr. The sodium/naphthalenesolution was added dropwise to the PEG solution, the solution wasstirred under argon for 4 hr. Ethyl bromoacetate (1.2 eq.) was thenadded and the solution was stirred under argon for 12 hr. The solutionwas filtered and the filtrate was precipitated in cold hexane, collectedby filtration and dried in vacuum oven for 24 hr. The dried substancewas dissolved in deionized water followed by addition of sodiumhydroxide (1 eq.). The solution was stirred at 40° C. for two hr,extracted by methylene chloride for two times and evaporated by rotaryevaporation.

2.6. To synthesize α-hydroxy, ω-glutarate PEG, PEG (1 eq.) was dissolvedin dry THF followed by addition of glutaric anhydride (1.5 eq.) andglutaric acid (0.001 eq.). The solution was stirred at 70° C. for 48 hr,cooled down, precipitated in cold hexane, collected by filtration anddried in vacuum oven for 24 hr.

2.7. To synthesize α-hydroxy, ω-triethoxysilane PEG, α-hydroxy,ω-acrylate PEG (1 eq.) from series 2.8 was dissolved in dry THF followedby addition of triehyoxysilane (5 eq.) and chloroplatinic acid (agrain). The solution was stirred at 60° C. for 48 hr, cooled down,precipitated in cold hexane, collected by filtration and dried in vacuumoven for 24 hr.

2.8. To synthesize α-hydroxy, ω-acrylate PEG, PEG (1 eq.) was dissolvedin dry THF followed by the addition of acryloyl chloride (1.5 eq.) andtriethylamine (1.7 eq.). The solution was stirred at room temperaturefor 2 hr, filtered and the filtrate was precipitated in cold hexane,collected by filtration and dried in vacuum oven for 24 hr.

2.9. To synthesize α-hydroxy, ω-aldehyde PEG, PEG (1 eq.) was dissolvedin DMSO and the solution was added dropwise to the acetic anhydride (20eq.) and stirred at room temperature for 2 hr. Ether was then added tothe solution and stirred for 5 min at room temperature and placed in the−20° C. freezer for 5 minutes to precipitate. The precipitate wascollected by filtration and then dissolved in minimal amounts ofmethylene chloride and reprecipitated likewise twice by ether. Theprecipitate was dried in vacuum oven for 24 hr.

2.10. To synthesize α-hydroxy, ω-halo PEG, PEG (1 eq.) was dissolved intoluene followed by addition of triethylamine (1.2 eq.). The solutionwas stirred at 60° C. for 30 min followed by addition of thionyl bromide(1.2 eq.) and stirred at 60° C. for 1 hr. The hot solution was filteredthrough celite and the filtrate was kept in refrigerator at −4° C. for24 hr. The precipitate was collected by filtration and dried in vacuumoven for 24 hr.

2.11. To synthesize α-hydroxy, ω-succinimidylglutrate PEG, α-hydroxy,ω-glutarate PEG (1 eq.) resulted from series 2.6 anddicyclohexylcarbodiimide (DCC 1.2 eq.) were dissolved in dry THFrespectively. N-hydroxy succinimide (1.2 eq.) was added to the PEGsolution followed by dropwise addition of DCC solution. The mixturesolution was stirred at room temperature for 6 hr, filtered and thefiltrate was precipitated in cold hexane, collected by filtration anddried in vacuum oven for 3 days and then stored under argon at −4° C. inthe refrigerator.

2.12. To synthesize α-hydroxy, ω-tryptophanylglutrate PEG, a-hydroxy,w-succinimidylglutrate PEG (1 eq.) from series 2.11 was dissolved in DMFfollowed by addition of tryptophan (1.5 eq.). The solution was stirredunder argon for 24 hrs, dialyzed in deionized water and dried bylyophilizer for 3 days.Series 3: Alpha-Acrylate Heterobifunctional PEG Derivatives:

3.1. To synthesize α-acylate, ω-tosyl PEG, α-hydroxy, ω-tosyl PEG (1eq.) from series 2.2 was dissolved in dry THF followed by addition ofacryloyl chloride (2 eq.) and triethylamine (2.2 eq.). The solution wasstirred at room temperature for 2 hr, filtered and the filtrate wasprecipitated in cold hexane, collected by filtration and dried in vacuumoven for 24 hr.

3.2. To synthesize α-acylate, ω-phthalimidyl PEG, α-acylate, ω-tosyl PEG(1 eq.) from series 3.1 and potassium phthalimide (2 eq.) were dissolvedin toluene and stirred at 50° C. for 20 hr. The solution was cooleddown, filtered and the filtrate was precipitated in cold hexane,collected by filtration and dried in vacuum oven for 24 hr.

3.3. To synthesize α-acylate, ω-cyanoalkyl PEGs, α-hydroxy, ω-acrylatePEG (1 eq.) from series 2.8 was dissolved in dry methylene chloridesolution followed by the addition of fine sodium metal (1.5 eq.) andstirred for 12 hr at room temperature. Excess amount of acrylonitrilewas added to the solution, stirred for 12 hr, filtered, and dried byrotary evaporation.

3.4. To synthesize α-acylate, ω-carboxy PEG, α-hydroxy, ω-carboxy PEG (1eq.) from series 2.5 was dissolved in dry THF followed by addition ofacryloyl chloride (1.2 eq.) and triethylamine (1.5 eq.). The solutionwas stirred at room temperature for 2 hr, filtered and the filtrate wasprecipitated in cold hexane, collected by filtration and dried in vacuumoven for 24 hr.

3.5. To synthesize α-acylate, ω-acryloylcarboxy PEG, ω-carboxy PEG (1eq.) from series 2.5 was dissolved in dry THF followed by addition ofacryloyl chloride (3 eq.) and triethylamine (3.5 eq.). The solution wasstirred at room temperature for 2 hr, filtered and the filtrate wasprecipitated in cold hexane, collected by filtration and dried in vacuumoven for 24 hr.

3.6. To synthesize α-acylate, ω-glutarate PEG, α-hydroxy, ω-glutaratePEG (1 eq.) from series 2.6 was dissolved in dry THF followed byaddition of acryloyl chloride (1.2 eq.) and triethylamine (1.5 eq.). Thesolution was stirred at room temperature for 2 hr, filtered and thefiltrate was precipitated in cold hexane, collected by filtration anddried in vacuum oven for 24 hr.

3.7. To synthesize α-acylate, ω-acryloylglutarate PEG, α-hydroxy,ω-glutarate PEG (1 eq.) from series 2.6 was dissolved in dry THFfollowed by addition of acryloyl chloride (3 eq.) and triethylamine (3.5eq.). The solution was stirred at room temperature for 2 hr, filteredand the filtrate was precipitated in cold hexane, collected byfiltration and dried in vacuum oven for 24 hr.

3.8. To synthesize α-acrylate, ω-acrylate PEG, PEG (1 eq.) was dissolvedin dry THF followed by the addition of acryloyl chloride (3 eq.) andtriethylamine (3.5 eq.). The solution was stirred at room temperaturefor 2 hr, filtered and the filtrate was precipitated in cold hexane,collected by filtration and dried in vacuum oven for 24 hr.

3.9. To synthesize α-acylate, ω-triethoxysilane PEG, α-hydroxy,ω-triethoxysilane PEG (1 eq.) from series 2.7 was dissolved in dry THFfollowed by the addition of acryloyl chloride (1.2 eq.) andtriethylamine (1.5 eq.). The solution was stirred at room temperaturefor 2 hr, filtered and the filtrate was precipitated in cold hexane,collected by filtration and dried in vacuum oven for 24 hr.

3.10. To synthesize α-acylate, ω-aldehyde PEG, α-hydroxy, ω-aldehyde PEG(1 eq.) from series 2.9 was dissolved in dry TUF followed by theaddition of acryloyl chloride (1.2 eq.) and triethylamine (1.5 eq.). Thesolution was stirred at room temperature for 2 hr, filtered and thefiltrate was precipitated in cold hexane, collected by filtration anddried in vacuum oven for 24 hr.

3.11. To synthesize α-acylate, ω-halo PEG, α-hydroxy, ω-halo PEG (1 eq.)from series 2.10 was dissolved in dry THF followed by the addition ofacryloyl chloride (1.2 eq.) and triethylamine (1.5 eq.). The solutionwas stirred at room temperature for 2 hr, filtered and the filtrate wasprecipitated in cold hexane, collected by filtration and dried in vacuumoven for 24 hr.

3.12. To synthesize α-acylate, (o-succinimidylglutrate PEG, α-hydroxy,ω-succinimidylglutrate PEG (1 eq.) from series 2.11 was dissolved in dryTHF followed by the addition of acryloyl chloride (1.2 eq.) andtriethylamine (1.5 eq.). The solution was stirred at room temperaturefor 2 hr, filtered and the filtrate was precipitated in cold hexane,collected by filtration and dried in vacuum oven for 24 hr.

3.13. To synthesize α-acylate, ω-succinimidylglutrate PEG, α-hydroxy,ω-tryptophanylglutrate PEG (1 eq.) from series 2.12 was dissolved in dryTHF followed by the addition of acryloyl chloride (1.2 eq.) andtriethylamine (1.5 eq.). The solution was stirred at room temperaturefor 2 hr, filtered and the filtrate was precipitated in cold hexane,collected by filtration and dried in vacuum oven for 24 hr.

Series 4: Homo-Bifunctional PEG Derivatives: Modified PEGS wherein the αand the ω termini have the same functional groups can also be fabricatedusing the same approach. Thus, using the chemistries described herein,bis-acrylate, bis-tosylate, bis-phthalimidyl, bis-cyanoalkyl,bis-carboxylate, bis-acryloylcarboxylate, bis-glutarate,bis-acryloylglutarate, bis-trialkoxysilane, bis-aldehyde,bis-N-succinimidyl, and bis-tryptophanylglutarate derivatives can befabricated.

Thus, according to the present invention, a polymer matrix, preferablygelatin, is modified to contain one or more of the modified PEGmolecules disclosed herein. The PEG molecule may be bis-modified, usingthe same type of moiety. Or, the α-terminus of the PEG may have adifferent moiety than the ω-terminus. Both versions of the modified PEGmolecules, as incorporated into a hydrogel, fall within the scope of thepresent invention.

Interpenetrating Network Hydrogels (IPNs):

The above described PEG-modified hydrogels can also be used as a firstpolymer matrix in an interpenetrating network of two distinct polymermatrices. In this aspect of the invention, the PEG-modified hydrogels asdescribed above are admixed with a polymerizable mixture of monomers. Apolymerization reaction is then initiated, whereby the mixture ofmonomers polymerizes in situ, thereby forming a second polymer matrixthat interpenetrates with the first polymer matrix.

It is much preferred that the plurality of monomers that forms thesecond polymer matrix is polymerizable by a means other than chemicalinitiation. Chemically polymerizable monomers are, however, within thescope of the invention. In the preferred embodiment, the monomers arephotopolymerizable. Thus, the monomers are admixed with the firstpolymer matrix. The mixture is then exposed to a suitable wavelength ofradiation (e.g., infrared, visible, or ultraviolet) that will result ina photo-initiated polymerization reaction. The source for the radiationcan be any source that generates radiation of the required wavelength,such as lamps (incandescent, fluorescent, ion discharge, etc.), lasers(CO₂, Ne—Ne, etc.), and light-emitting diodes.

The preferred photopolymerizable monomers are acrylates, diacrylates,and poly(acrylates) (including PEG-acrylates, PEG-diacrylates, andTMPTA), acrylic acid, and acryloyl halides, such as acryloyl chloride,and mixtures thereof. When a plurality of different monomers is admixedwith the first polymer matrix, the polymerization reaction will, ofcourse, result in the second polymer matrix being a co-polymer. Thus,the second polymer matrix may comprise a homo-polymer matrix or aco-polymer matrix of any description (e.g., alternating, block, or graftco-polymers).

FIG. 6 is a schematic representation of interpenetrating networkhydrogels according to the present invention. The gels can containliving cells or pharmcalogically-active agents, or both.

EXAMPLES

The following Examples are included herein solely to provide a morecomplete and consistent understanding of the invention disclosed andclaimed herein. The Examples do not limit the scope of the invention inany fashion.

Example 1 Synthesis and Characterization of Heterobifunctional PEGs

All reagents were purchased from Sigma-Aldrich (St. Louis, Mo.) unlessstated otherwise. A summary of the chemical reactions and structure ofcritical intermediates and final products is presented in FIG. 1.

To synthesize α-methyl-ω-acrylate PEGs (M-PEG), monomethoxy PEGs (2 kDaor 5 kDa, purchased from Fluka, a division of Sigma-Aldrich) weredissolved in dry tetrahydrofuran (THF) solution followed by the additionof triethylamine (TEA, 2 eq.) and acryloyl chloride (AC, 4 eq.)⁽¹⁴⁾ atroom temperature under Ar for 10 min, filtered, dried by rotaryevaporation, re-dissolved in CH₂Cl₂, and precipitated in cold hexane.The final product was filtered, dried, and stored in vacuo at roomtemperature.

To synthesize α-cyanoethyl-ω-acrylate-PEGs (CN-PEG), PEG-diols (2 kDa or5 kDa) (1 eq.) were dissolved in dry CH₂Cl₂ solution followed by theaddition of fine sodium metal (2 eq.) stirred for 12 hr at roomtemperature. An excess amount of acrylonitrile was added into thesolution^((15,16)), stirred for 12 hr, filtered, and dried by rotaryevaporation. The product thus formed (i.e., α-nitrile-ω-hydroxy-PEG) wasdissolved in dry THF, followed by the addition of TEA (2 eq.) and AC (4eq.). The solution was stirred under Ar for 10 min at room temperature.Triethylammonium chloride was removed by filtration and the solvent wasremoved by rotary evaporation. The final product was re-dissolved intoCH₂Cl₂, precipitated in cold hexane, filtered, and stored in vacuo atroom temperature.

To synthesize α-carboxyl-ω-acrylate-PEGs (COOH-PEG), sodium (3.5 eq) inmineral oil was dried, dissolved in dried THF with naphthalene (3.5 eq),and stirred for 1 hr under Ar at room temperature.⁽¹⁷⁾ Thesodium/naphthalene solution thus formed was added drop wise intoPEG-diols (2 kDa or 5 kDa) (1 eq.) dissolved in dried THF under Ar for 4hr. Ethyl bromoacetate (4 eq) was added to the ionized PEG solution,stirred for 12 hr, filtered, precipitated in cold hexane, andre-dissolved in distilled water (1 eq) with sodium hydroxide (3 eq)⁽¹⁸⁾,followed by reflux for 24 hr at room temperature. Solvent was removed byrotary evaporation and the solid was re-dissolved in CH₂Cl₂, filtered,precipitated in cold hexane, dried in vacuo. The solid of mainlyα-carboxyl-ω-hydroxyl-PEGs (1 eq.) was dissolved in dried THF followedby the addition of TEA (2 eq.) and AC (4 eq), stirred at roomtemperature for 10 min under Ar, filtered, precipitated in cold hexane,filtered, dried, and stored in vacuo at room temperature.

To synthesize α-phthalimide-ω-acrylate-PEGs (PT-PEG), PEG-diols (2 kDaor 5 kDa) (1 eq.) were dissolved in dry CH₂Cl₂ solution followed by theaddition of TEA (4 eq.) and p-toluenesulfonyl chloride (2 eq.)⁽¹⁹⁾ andstirred under Ar for 8 hr at room temperature. Solvent was removed byrotary evaporation to obtain yellowish white solids. This mixture ofPEG-diols, α-hydroxyl-ω-tosyl-PEGs, and bis-tosyl-PEG (1 eq.) wasdissolved in dry THF, followed by the addition of TEA (2 eq.) and AC (4eq.), stirred at room temperature under Ar for 10 min, filtered toremove triethylammonium chloride, dried via rotary evaporation to removesolvents, re-dissolved into CH₂Cl₂, and precipitated in cold hexane. Thesolid product (mainly α-tosyl-ω-acrylate-PEG) was filtered, dried invacuo, dissolved (1 eq.) in CH₂Cl₂, followed by the addition ofpotassium phthalimide (3 eq.)⁽²⁰⁾ and refluxed for 18 hr. The solutionwas filtered, dried via rotary evaporation to remove solvents,re-dissolved into CH₂Cl₂, precipitated in cold hexane, filtered, dried,and stored in vacuo at room temperature.

All intermediates and final products were analyzed with ¹H- and ¹³C-NMRwith samples dissolved in CDCl₃ and with a reverse-phase HPLC system(10% to 100% acetonitrile at a flow rate of 1 ml/min in 60 min with aJordi 500 Å column on a Gilson system) coupled to an automatedmulti-sample sampler-fraction collector. Detectors included UV/Vis (200and 254 nm), photodiode array, and evaporative light scatteringdetectors.

The above-described heterobifunctional PEGs (hPEGs) were employed as acomponent in the formation of hydrogels to investigate the influence ofhPEG concentration, molecular weight, and terminal moiety on the surfacehydrophilicity and cell interaction. The hPEGs were utilized in thehydrogel formulation following procedures described hereinabove. Seealso references (10-13). The network thus formed is a random copolymerof Ac, TMPTA, and hPEG, with, for example, an acrylate ω-terminal and anα-terminal of a different chemical moiety (XPEGmA).

Specifically, XPEGmAs were grafted to a gelatin polymer matrix withvarious dangling terminal functional groups and incorporated throughoutthe polymer matrix by copolymerizing the acrylate terminal into arandomly polymerized network of Ac and TMPTA.⁽¹⁰⁻¹³⁾ This type ofpolymer network containing M-PEG is nonionic, low swelling, glassy whendry, optically transparent, and colorless.⁽¹⁰⁻¹³⁾ In spite of therelatively high mass fraction of M-PEGs present, minimal swelling wasobserved for the polymer due to the highly cross-linked and hydrophobicnature of the TMPTA network. Differential scanning calorimetry analysisshowed that these materials are completely amorphous and the M-PEGcomponent is completely phase-mixed in the cross-linked TMPTAmatrix.⁽¹⁰⁾

The surface hydrophilicity of XPEGmA-co-Ac-co-TMPTA networks wasquantified with an underwater air bubble captive system. The hydrogelwas completely suspended in water that was maintained at aphysiologically-relevant temperature of 37.5° C. An air bubble wasplaced at the down side of the gel and the contact angle was measuredusing a modified computer-assisted video contact angle system (AST Inc).Measurement was made at six randomly selected areas, averaged, andrepeated three times on three different polymer samples (n=3). Becausethe air bubble contact angle was measured through the aqueous phase andperformed under water, the value obtained is essentially thewater-receding contact angle; furthermore, the higher the contact angle,the higher is the hydrophilicity of the film.

The gels so formed were then evaluated for their interaction withcultured cells. Human neonatal dermal fibroblasts at a concentration of75,000 per 1 ml of Fibroblast Basal Medium with human fibroblast growthfactor-b (0.5 mg/ml), insulin (0.5 mg/ml), and 5% fetal bovine serum(Clonetics, San Diego, Calif.) were incubated with theXPEGmA-co-Ac-co-TMPTA network. At 2, 24, and 48 hr thereafter, adherentcell morphology and density were manually quantified using acomputer-assisted video analysis system coupled to an inverted lightmicroscope.

All experimental results are expressed in mean±standard deviation(S.D.).

Each sample was independently repeated three times (n=3). Comparativeanalyses were performed with Statview® 4.5 using analysis of varianceand Fisher's protected least significant difference test at 95%confidence level (p<0.05).

¹³C-NMR chemical shifts for M-PEG, CN-PEG, COOH-PEG, and PT-PEGintermediates and final products synthesized from 2 kDa PEG precursorsare listed in Table 1. TABLE I ^(—)C-NMR chemical shifts (ppm) forM-PEG. CN-PEG, COOH-PEG. and PT-PEG critical intermediates and finalproducts synthesized from 2K Da PEG-diol precursors Chemical Shifts ofDesignated Carbon (in superscript) with Compounds with the FollowingGeneral Structure Chemical Group of n-CH₂ C^(α1) C^(β1) C^(α2) C^(β2)C⁽¹⁾ C⁽²⁾ C⁽³⁾ C⁽⁴⁾ C⁽⁵⁾ C⁽⁶⁾ C⁽⁷⁾ Terminal YY-C^((α1))H²C^((β1))H₂O(CH₂CH₂O)_(n)C^((β1))H₂C^((α2))H₂OH 70.4 61.372.4 61.3 72.4 — — — — — — — -OH 70.5 30.3 71.2 62.9 72.3 — — — — — — —-Br 70.4 19.8 66.2 62.3 72.4 119.0 — — — — — — -C⁽¹⁾N 70.4 68.2 70.261.3 72.4 53.7 171.2 — — — — — -OC⁽¹⁾H₂C⁽²⁾OOH 70.4 64.4 71.8 61.6 69.758.2 — — — — — — -OC⁽¹⁾H₃ 70.5 68.6 69.2 61.6 72.5 163.6 114.7 131.6130.0 21.8 — —

Y-C^((α1))H²C^((β1))H₂O(CH₂CH₂O)_(n)C^((β1))H₂OC⁽¹⁾OC⁽²⁾HC⁽³⁾H₂ 70.564.6 71.1 63.9 68.2 165.3 130.6 128.2 58.2 — — — -OC⁽⁴⁾H₃ 169.5 70.418.6 66.5 64.3 68.7 165.9 130.6 128.0 117.7 — — — -C⁽⁴⁾N 169.2 70.5 68.570.8 64.6 68.4 166.0 130.9 128.2 53.6 170.3 — — -OC⁽⁴⁾H₂C⁽⁵⁾OOH 169.570.5 37.2 67.8 170.6 63.8 68.8 167.8 130.9 128.2 168.0 132.1 123.1 133.9

For all samples, the methyl stretch and the “b” carbon of the PEG chainswere observed at approximately 68 to 72 ppm; whereas, the “a” carbonshift was highly dependent on the terminal group (Y). For compounds witha general structure of HOCH₂CH₂O(CH₂CH₂O)_(a)CH₂CH₂—Y, where Y is —OH,—Br, —CN, —OCH₂COOH, —OCH₃, or tosyl group, the assigned carbon showedsignals at the corresponding chemical shift. For the final acrylatedproduct with a general chemical structure ofX—CH₂CH₂O(CH₂CH₂O)_(a)CH₂CH₂OCOCHCH₂, where X is: —OCH₃, —CN, —OCH₂COOH,or phthalimide, three unique chemical shifts were observed thatcorrespond to the three carbons of the acrylate group. Specifically, thechemical shifts for —COO— stretch and —CHCH₂ stretch were observed at165.3 to 170.6 and 128.0 to 130.9 ppm, respectively. In addition,appropriate chemical shifts were observed for the assigned carbon foreach terminal group (Y). Similar NMR results were obtained when 5 kDaPEGs were utilized in lieu of 2 kDa PEGs as precursors in the synthesisscheme for all compounds shown in Table 1.

To determine percent conversions of the final product of M-PEG, CN-PEG,COOH-PEG, and PT-PEG, HPLC analyses performed (Table 2 and FIGS. 2A and2B) from various HPLC detectors were utilized to elucidate the chemicalstructure of each individual peak of a given chromatogram. In addition,each fraction was collected with an automated fraction collector andre-analyzed using ¹H and ¹³C NMR to ascertain further the chemicalcomposition(s) of each collected fraction. Results showed 100%conversion for M-PEG from the PEG starting material. CN-PEG showedapproximately 65% conversion with no other acrylated side products.PT-PEG showed an approximate 65% conversion with about 5% of the finalproduct containing another acrylate side-product (e.g.,α-tosyl-ω-acrylate-PEG). COOH-PEG showed an approximate 60% conversionwith an additional 10% of other acrylated side-products (e.g.,α-hydroxyl-ω-acrylate-PEG and bis-acrylate-PEG). TABLE 2 Comparison ofHPLC retention time, normalized peak area, and percent conversion forM-PEG, CN-PEG, COOH-PEG, and PT-PEG synthesized from 2K Da PEG-diolprecursors PEG Retention Normalized Conversion UV PEG Derivative ProductTime (min) Peak Area Factor (%) Signal Identification PEG-diol 16 1.0 1no α-melhyl-ω-hydroxyl M-PEG 21 1.0 100 strong α-methyl-ω-acrylateCN-PEG 21 1.1 13 strong bis-ethylcyano 23 5.2 63 strongα-ethylcyano-ω-acrylate 24 2.0 24 no α-nitrile-ω-hydroxy COOH-PEG 11 2.514 no bis-carboxyl 13 2.3 13 no α-carboxyl-ω-hydroxyl 15 10.1 57 weakα-carboxyl-ω-acrylate 16 1 6 no bis-hydroxyl 19 1.2 7 weakα-hydroxyl-ω-acrylate 23 0.6 3 weak bis-acrylate PT-PEG 22 2.0 7 weakα-tosyl-ω-acrylate 24 8.1 26 strong bis-phthalimide 26 19.5 64 strongα-phthalimide-ω-acrylate

These results validate the synthesis of the XPEGmAs that were employedas a main component in the hydrogel synthesis. Based on the gelsynthesis scheme, hPEG containing one or more acrylate groups will becovalently incorporated into the network; whereas, those without anyacrylate groups will be removed from the network after the equilibrationstep in water as a part of the network formation procedure. Although thefinal product of each XPEGmA was not further purified prior to thepolymer synthesis, the low concentration of other acrylated sideproducts plays a minimal role in the network composition.

These heterobifunctional intermediates and final products of XPEGmA arestable under storage in vacuo at room temperature and can be modifiedfurther by a broad range of chemical methods for various applications.For example, the phthalimide group is a good protecting group that canbe hydrolyzed to form primary amines.

A previously developed polymer network formulation was adopted toelucidate the effect of the PEG chemistry on the surface characteristicsof the resulting hydrogels. Polymer networks containing various XPEGmAsat several concentrations and different molecular weights weretransparent or translucent. The network surface hydrophilicity wasquantified using an under water contact angle system and was found to bedependent of three factors: the molecular weight of the startingmaterial PEG, the dangling terminal functional group, and theconcentration of the XPEGmA in the network (see Table 3 and FIGS. 3A and3B). TABLE 3 Surface hydrophilicity of the XPEGmA-co-Ac-co-TMPTA networkcontaining XPEGmA of various concentration, molecular weight, andterminal moiety XPEGmA XPEGmA concentration in the network formulation(g/ml) type 0.2 0.4 0.8 1.25 2.5 2K (Da) M-PEG 37 ± 8 34 ± 6 37 ± 4 34 ±2 29 ± 5 CN-PEG 46 ± 4 32 ± 2 † 36 ± 5 † 37 ± 2 † 39 ± 2 †§ COOH-PEG 44± 3 42 ± 6 38 ± 6 † 46 ± 7 § 43 ± 4 § PT-PEG 23 ± 4 § 45 ± 4 †§ 40 ± 6 †38 ± 2 † 41 ± 2 †§ 5K (Da) M-PEG 41 ± 6 45 ± 6 ‡ 51 ± 7 ‡ 42 ± 5 ‡ 47 ±1 ‡ CN-PEG 46 ± 5 32 ± 2 †§ 36 ± 7 †§ 37 ± 1 †§ 39 ± 3 †§ COOH-PEG 51 ±3 ‡§ 42 ± 2 † 39 ± 1 †§ 43 ± 4 † 44 ± 4 † PT-PEG 46 ± 4 ‡ 46 ± 1 51 ± 740 ± 3 † 39 ± 2 †§

First, when a given concentration of XPEGmA containing a given danglingterminal group in the network was considered, an increase in themolecular weight of the terminal group significantly lowered thehydrophilicity of networks containing M-PEG (0.4 to 2.5 g/ml), COOH-PEG(0.2 g/ml), or PT-PEG (0.2 g/ml). For other networks, the molecularweight of XPEGmA did not significantly affect the hydrophilicity.

Second, the different terminal moiety of XPEGmA showed a variable effecton the surface hydrophilicity when compared with that of M-PEG of givenmolecular weight and concentration.

Third, the XPEGmA concentration in the network formulation showedvarious correlations with hydrophilicity. Because XPEGmA was employed inthe network formation without further purification, the potential effectof differential percent conversion of acrylated hPEG on surfacehydrophilicity must be addressed. M-PEG showed a 100% conversion and thenetwork containing M-PEG demonstrated no changes in surfacehydrophilicity with increasing M-PEG concentration. Whereas for otherXPEGmAs, various correlations among hydrophilicity and the type, percentconversion (ca. 60 to 100%), and concentration were observed. Hence, itwas concluded that the percent conversion of XPEGmA within 60 to 100%did not affect the dependency of XPEGmA concentrations onhydrophilicity. These analyses determined that the network surfacehydrophilicity was predominately influenced by the XPEGmA concentrationin the network formulation with the molecular weight and the terminalmoiety playing lesser roles.

Next, XPEGmA-co-Ac-co-TMPTA networks containing various XPEGmAs atseveral concentrations were employed to determine the effect of surfacecharacteristics of the gel on human fibroblast adhesion. All adherentcells showed extensive pseudopodial extension and cytoplasmic spreading,with some cells exhibiting polar cell body morphology. The results (seeTable 4) showed that adherent cell density was primarily dependent onthe XPEGmA concentration in the network formulation. Specifically,adherent cell density decreased with increasing XPEGmA concentration atall culture time. No adherent cell was observed on networks containingXPEGmA concentration between 1.25 to 2.5 g/ml at all culture times.These trends were independent of the XPEGmA molecular weight andterminal moiety. No direct mechanistic correlation can be made betweennetwork surface hydrophilicity and adherent cell density because severalinterrelated complex parameters (e.g., XPEGmA chemicophysicalproperties, adsorption of serum adhesion-mediating proteins, etc.)contribute to these two phenomena. However, the adherent cell densitydecreased with increasing XPEGmA concentration for all samples. TABLE 4Adherent human dermal fibroblast density on the XPEGmA-co-Ac-co-TMPTAnetwork containing XPEGmA of various concentration, molecular weight,and terminal moiety Culture Time (hr) 2 hr 24 hr 48 hr XPEGmA XPEGmAconcentration in the network formulation (g/ml) type 0.2 0.4 0.8 1.252.5 0.2 0.4 0.8 1.25 2.5 0.2 0.4 0.8 1.25 2.5 2K (Da) M-PEG 3 ± 2 3 ± 20 0 0 5 ± 2 2 ± 1 0 0 0 3 ± 2 4 ± 2 1 ± 1 0 0 CN-PEG 5 ± 4 2 ± 1 1 ± 1 00 3 ± 2 4 ± 3 1 ± 1 1 ± 1 0  5 ± 34 4 ± 3 3 ± 1 5 ± 2 0 COOH-PEG 2 ± 1 1± 0 1 ± 1 0 0 3 ± 1 2 ± 1 6 ± 4 0 ± 0 0 3 ± 2 1 ± 1 1 ± 1 1 ± 1 0 PT-PEG2 ± 1 2 ± 1 1 ± 1 1 ± 0 0 1 ± 0 3 ± 2 3 ± 2 0 0 3 ± 1 2 ± 1 3 ± 3 0 ± 00 5K (Da) M-PEG 3 ± 1 3 ± 2 0 0 0 3 ± 3 3 ± 2 1 ± 1 0 0 2 ± 1 3 ± 2 1 ±1 0 0 CN-PEG 2 ± 2 1 ± 1 1 ± 0 0 0 4 ± 3 5 ± 4 0 ± 0 2 ± 1 0 3 ± 2 3 ± 20 ± 0 2 ± 2 0 PT-PEG 3 ± 1 1 ± 0 0 ± 0 0 0 2 ± 1 2 ± 2 0 ± 0 0 0 3 ± 1 3± 2 1 ± 1 0 0 COOH-PEG 4 ± 0 1 ± 1 1 ± 0 0 0 4 ± 2 2 ± 1 1 ± 0 1 ± 0 0 2± 1 3 ± 1 1 ± 0 0 ± 0 0All values are expressed in ×100 cells/mm² (rounded-off for clarity,mean ± S.D., n = 3).

The results of this Example show that the presence of two distinctchemical moieties (i.e., carboxylic acids of the poly-acrylic acidbackbone and the distinct functional group at the dangling terminus ofXPEGmA grafted at the pendent chain configuration) within the hydrogelscan be employed to bind (covalently) two or more distinct types ofbiofunctional molecules such as peptides and pharmaceutics by employingdistinct chemical methodologies. Furthermore, the high content of PEGsin this system reduced protein adsorption and effectively eliminatednonspecific cell adhesion that would occur as a result, thus permittingthe modulating of cellular function mediated uniquely by the multipleimmobilized biofunctional agent (10-13). The invention thus providesmulti-functional hydrogels that can be used, for example, to studycomplex biological systems and to deliver therapeutic agents locally andsystemically.

Example 2 Drug Release Kinetics

This Example explores the swelling and drug release kinetics of variousgelatin-based hydrogels. The hydrogels were cross-linked by variousmeans, and contained various modifications of the gelatin backbone. Theeffect of pH on the drug release kinetics of these gels was alsoinvestigated.

As noted above, cross-linking gelatin produces a hydrogel of highmolecular weight and reduces or prevents gelatin dissolution. Thecross-linking agents used in this Example were: 0.1%, 0.01%, and 0.001%(v/v) glutaraldehyde aqueous solutions, and self-cross-linking vialiquid nitrogen immersion followed by baking. The backbone modificationsto the gelatin were the addition of polyethylene glycol (PEG) orethylenediaminetetraacetic dianhydride (EDTAD) or both. PEG has lowimmunogenicity and cytotoxicity. EDTAD has low toxicity and the lysylresidues of gelatin can be modified with EDTAD in a relatively fastreaction following facile procedures. See Hwang & Damodaran (1996) J.Agric. Food. Chem. 44:751-758. Also, modifying gelatin with EDTADintroduces polyanionic molecules into the gelatin chain, therebyimproving the swelling capability of the gelatin hydrogels. The pHsinvestigated in this Example were pH 4.5, pH 7.0 and pH 7.4. Based onthe swelling/degradation and drug release kinetics of these hydrogelsunder the stated conditions anid in vivo analysis, these- hydrogels aresuitable as support matrices for the regeneration of rat neutral stemcells and as a drug carrier in mediating inflammation in vivo.

PEG diol (Aldrich, M_(n) 2 kD) was converted to PEG dialdehyde (PEGdial)by reacting PEG with acetic anhydride in DMSO in a molar ratio of1:80:140 for 4 hours at 25° C. The composition of PEG dialdehyde wasconfirmed using the reverse-phase HPLC system and parameters asdescribed in Example 1. This reaction produces a mixed product of PEGmonoaldehyde and PEG dialdehyde. PEG dialdehyde had an elution time ofapproximately 11.5 min. and was approximately 80 wt % of the finalproduct.

The lysyl amino groups of gelatin samples (Sigma, St. Louis, Mo.; TypeA, from porcine skin, 300 bloom, cell culture tested) were modified byPEGdial to form PEG-modified gelatin (PG). Gelatin samples were alsomodified using EDTAD (Aldrich) to form EDTAD-modified gelatin (EG).Still further gelatin samples were modified with PEGdial and EDTAD toyield PEG-modified-EDTAD-modified gelatin (P/EG). PG or P/EG was createdby adding PEGdial dissolved in 10 ml of H₂O (Milli-Q synthesis, 18.2MΩ-cm, Millipore) and NaCNBH₃ dissolved in 10 ml of H₂O separately andsimultaneously to a 5% (w.v) gelatin or EG solution at 50 to 60° C. for24 hours in a wt ratio of gelatin/EG: PEGdial: NaCNBH₃ of(1:0.66:0.186). The theoretical maximum percent modification using thismethod is 100% modification of gelatin lysyl residues, based on anaverage 300 bloom gelatin molecular weight and average lysine content ofthe gelatin. See, e.g., Merck Index, 12^(th) Ed. (1996) #4388, p. 742.EG was created by adding EDTAD to a 1% (w/v) gelatin solution at pH 10,40° C. for 3 hours in a wt ratio of gelatin:EDTAD of 1:0.034. Thetheoretical maximum percent modification of gelatin lysyl residues usingthis method is 38%. Thus, modifications larger than this indicate thatboth functional groups of the added EDTAD have bonded to lysyl residuesin the gelatin, thereby cross-linking the gelatin chains. The level ofgelatin modification was quantified using the 2,4,6-trinitrobenzenesulfonic acid spectrophotometric method. See Hwang & Damodaran, supra,and Offner & Bubnis (1996) Pharm. Res. 13:1821-1827.

To make the hydrogels, 10% (w/v in H₂O) solutions of gelatin (G), 10%PG, 40% EG and 60% P/EG were heated to approximately 70° C. and pouredinto petri dishes (60×15 mm, Cole-Parmer) to a thickness of 6 mm andallowed to set at room temperature overnight. Hydrogels were cut into 1cm diameter circular discs or into 0.5×0.5 cm squares, and cross-linkedwith 0.1, 0.01 or 0.001% (v/v in H₂O) gluteraldehyde (ElectronMicroscopy Sciences, EM grade, 10% (v/v) aqueous solution) for 6 hourswith gentle shaking. Cross-linked hydrogels were washed with H₂O tentimes for 3-5 min. Washed hydrogels were left overnight in H₂O forcontinued leaching of the gluteraldehyde. Hydrogels were then dried atroom temperature in ambient air for 48 hours and weighed. Separately,hydrogels of 10% by wt gelatin were dried in ambient air for 48 hours,frozen in liquid nitrogen for 30 seconds to 1 minute and then baked at130-135° C. for 8.5 hours (self-cross linked; LN₂-baked G). Not allhydrogel formulations withstood the cross-linking, washing and dryingsteps, mainly due to hydrolysis. The hydrogel formulations that wereincluded in the swelling/degradation and in vitro drug release studieswere the 0.1% glutaraldehyde cross-linked G, PG, EG, and P/EG gels; the0.01% glutaraldehyde cross-linked G, PG, and EG gels; the 0.001%glutaraldehyde cross-linked G and PG gels; and the self-cross linkedLN₂-baked G. Swelling study results for P/EG hydrogels and in vitro andin vivo drug release studies are ongoing and results are not includedhere.

For in vitro drug release studies, each hydrogel was loaded withchlorhexidine digluconate (CHD; Sigma, 20% (w/v) aqueous solution) usingthe same drug loading density used for dexamethasone in the in vivostudies (150 μg/kg/day, dosage of 21 d). Assuming a rat weight of 0.2kg, this loading density is equivalent to 630 μg/hydrogel. Based on themaximum swelling weight ratios from the swelling studies, each hydrogelwas loaded with 35 μL of CHD (18 mg/ml), a volume well below the maximumvolume the hydrogel could absorb. Hydrogels (0.5×0.5×0.6 cm) were placedinto individual wells in a 48-well tissue culture plate. CHD was addedto each well, and the hydrogels were allowed to absorb the drug solutionovernight (approximately 15 hours) with gentle shaking.

To evaluate swelling and degradation kinetics, dried hydrogels wereplaced in 5 ml of aqueous solutions of pH 4.5, pH 7.0 or pH 7.4 in awater bath at 37° C. Aqueous solutions were created by adjusting the pHof H₂O with dilute HCl and NaOH. Hydrogels were transferred to freshaqueous solutions at approximately 3 and 6 wks. Swollen hydrogels wereweighed at 2, 4, and 6 hours, 1, 2, 3, 4, and 5 days, and 1, 2, 3, 4, 5,6, 7, and 8 weeks to characterize the swelling/degradation kinetics.Extreme care was taken to preserve the integrity of the hydrogels atevery step in the weighing process. The swelling weight ratio at eachtime point for each hydrogel was calculated as: (W_(s)−W_(d))/W_(d),where W_(s) is the weight of the swollen gel and W_(d) is the weight ofthe dry gel (in grams). The maximum swelling weight ratio that occurredover 8 weeks and the time it occurred was also calculated (R_(max) &T_(max) respectively). The last attainable swelling weight ratio (due tohydrogel dissolution) and the time it occurred was also calculated(R_(fail) & T_(fail) respectively). Statistical analysis was performedusing ANOVA and Tukey multiple comparisons tests (p<0.05). Individualsample solutions from the swelling study were collected for ongoing GPCanalysis of degradation products (results not shown) (20% (v/v)acetonitrile: 0.1 M NaNO₃ at a flow rate of 0.7 ml/min, 60 min., usingthree Ultrahydrogel columns in series, Ultrahydrogel 250, 1000 andLinear, on a Waters system).

For in vivo studies, unmodified gelatin cross-linked in 0.1% and 0.01%gluteraldehyde were tested in vivo, following the established cageimplant system. See Kao & Anderson (1999) “Handbook of BiomaterialsEvaluation 2^(nd) ed., Taylor & Frances Publishing, Philadelphia, Pa.,pp. 659-671. Samples were placed inside a cylindrical cage (3.5 cmlong×1 cm diameter) constructed from medical grade stainless steel wiremesh. Empty cages were implanted as controls. All cages were implantedsubcutaneously in the back of 3-month-old female Sprague-Dawley rats. At4, 7, 14 and 21 days post-implantation, the inflammatory exudates thatcollected in the cages were withdrawn and analyzed for the quantitativeevaluation of cellular and humoral response to implantation usingstandard hematology techniques. The distributions of lymphocyte,monocyte, and polymorphonuclear leukocyte (PMN) subpopulations in theexudates were determined. Concurrently, the implanted materials wereretrieved for analysis of changes in the sample physiochemicalcomposition (e.g., percent mass loss).

Percent modification of the lysyl residues in gelatin by PEG and/orEDTAD was quantified using the TNBS method: The PG was found to be 10%modified, the EG 40% modified, and the P/EG 60% modified. All resultsreported here incorporate materials from the same batch of modifiedgelatin (i.e. 8% PG, 42% EG).

FIG. 5 is a graph depicting representative swelling/degradationkinetics. Time in hours is shown on the X-axis; swelling ratio is shownon the Y-axis. Key: G, 0.01% glutaraldehyde cross-linked=♦; 10% PG,0.01% glutaraldehyde cross-linked=▪; 40% EG, 0.01% glutaraldehydecross-linked=▴. Swelling/degradation studies showed that G modified withPEG significantly increased T_(max) and T_(fail), whereas G modifiedwith EDTAD significantly increased T_(max). Hydrogels cross-linked in0.01% or 0.001% gluteraldehyde showed a significant difference inT_(max) and T_(fail) over gels cross-linked in 0.1% gluteraldehyde. Thelevel of pH did not significantly affect R_(max), T_(max), R_(fail) andT_(fail). Table 5 shows R_(max), T_(max), R_(fail) and T_(fail) for alllevels of gluteraldehyde concentration, pH and gelatin backbonemodification. TABLE 5 R_(MAX), T_(MAX), R_(FAIL), AND T_(FAIL) FOR ALLLEVELS OF GLUTERALDEHYDE/HEAT TREATMENT, PH AND GELATIN BACKBONEMODIFICATION % gluteraldehyde fixation/heat G treatment pH Mod^(c) R-maxT-max R-fail T-fail 0.1% 4.5 G 6.30 108  4.11 >1344 PG 6.98 1344^(b) 6.98 >1344 EG 8.77 720  7.71 >1344 7.0 G 5.94 108  2.88 >1344 PG 6.641092  4.55 >1344 EG 12.04  1008  6.24 >1344 7.4 G 4.68 96 1.45  1092 PG6.60 1092  5.35 >1344 EG 894.17  924  892.52 >1344 0.01% 4.5 G 35.48  367.25  132 PG 11.54^(b) 24 5.80  420 EG 31.53   2 14.07   84 7.0 G 40.23 48 8.49   84 PG 10.63  96 8.36  336 EG 26.96   2 7.71  168 7.4 G 26.29 36 8.21   72 PG 10.48  96 5.06  336 EG 30.88  12 6.47  168 0.001% 4.5 G0.10  1 −0.01   2 PG 0   0 0   0 EG — — — — 7.0 G 0.33  1 0.17   2 PG 0  0 0   0 EG — — — — 7.4 G 0.36  1 0.36   1 PG 0   0 0   0 EG — — — —LN₂-baked G 4.5 G 3.96 24 2.06   252^(b) 7.0 G 4.76 24 0.40   72 7.4 G4.05 15 72   96^(a)All values expressed in mean (n = 2 − 3) with s.e.m. omitted forclarity.^(b)Significantly different from G under same experimental conditions;paired t-tests, p < 0.05.^(c)10% PG or 40% EG

In vivo studies following the cage implant system allowed the durationand magnitude of the host foreign body reaction to the implantedgelatin-based hydrogels (0.1% G and 0.01% G) to be evaluated. Thepresence of a high concentration (relative to control) ofpolymorphonuclear leukocytes (PMNs) in the exudates indicates an acuteinflammatory response, which occurs at the onset of implantation andattenuates with time. The presence of a high concentration (relative incontrol) of monocytes and lymphocytes in the exudates is indicative ofthe chronic inflammatory response. Thus, 0.1% G hydrogels elicited aslightly enhanced chronic inflammatory response at 7 days and anenhanced chronic inflammatory response at 14 days vs. the control andthat of 0.01% G. 0.01% G elicited a slightly enhanced chronicinflammatory response at 7 days vs. the control (see Table 6). By day21, all samples showed a comparable level of chronic inflammation vs.the controls the proceeded toward resolution. Percent mass loss ofsamples increased with increasing implantation time and was furtherincreased with decreasing percentage of gluteraldehyde fixation (resultsnot shown). TABLE 6 TOTAL AND DIFFERENTIAL LEUCOCYTE CONCENTRATION INTHE INFLAMMATORY EXUDATES OF GELATIN HYDROGELS CROSS-LINKED IN 0.1 OR0.01% GLUTERALDEHYDE Implantation Cell concentration (×cells/μL)^(a)Sample time (day) Total Lymphocyte Monocyte PMN Empty cage 4 184 ± 25 168 ± 23  16 ± 7  1 ± 1 (no sample) 7  57 ± 12^(c)  49 ± 10^(c) 7 ± 2 0± 0 14 55 ± 7  36 ± 3  12 ± 4  7 ± 5 21 91 ± 69 98 ± 54 20 ± 16 0 ± 00.1% 4 597 ± 392 255 ± 116 126 ± 113 217 ± 21  7 183 ± 129 78 ± 40 26 ±14 2 14  235 ± 65^(b) 118 ± 30^(b) 40 ± 16  79 ± 74^(b) 21 200 167 33 77 ± 750 0.01% 4 477 ± 195 412 ± 172 57 ± 28 8 ± 5 7 178 ± 78^(b) 157 ±80  17 ± 1^(b) 4 ± 3 14 72 ± 36 60 ± 29 10 ± 7  2 ± 1 21 93 ± 3  72 ± 5 9 ± 4 12 ± 11^(a)All values expressed in mean ± s.e.m. (n = 3 − 7).^(b)Represents p < 0.01 vs. respective values of “empty cage” controls.^(c)Represents p < 0.01 vs. respective values at day 4 of the samesample type.

This Example shows that gelatin backbone modifications and cross-linkingagent selection affect the swelling/degradative kinetics of modifiedgelatin-based hydrogels. By modulating these material properties andmonitoring how these changes affect drug release kinetics, anonimmunogenic, bioresorbable cell/drug carrier matrix can be made thatwill have desirable release characteristics based on such considerationsas the drug being used in the formulation, the length of the treatment,and the condition being treated, and the location of the implantedmatrix.

Example 3 In Vivo Modulation of Host Response Using Gels Grafted withFibronectin-Derived Biomimetic Oligopeptides

The host inflammatory reaction is a normal response to injury and thepresence of foreign objects. The magnitude and duration of theinflammatory process have a direct impact on biomaterial biostabilityand biocompatability. Thus, this Example investigates the performance ofgels fabricated according to the present invention that includefibronectin-derived biomimetic oligopeptides. Fibronectin in known toadsorb on a variety of biomaterials and play an important role in thehost-foreign body reaction. The RGD (SEQ. ID. NO: 1) and PHSRN (SEQ. ID.NO: 2) amino acid sequences are particularly interesting because thesesequences are present on adjacent loops of two connecting FIII modulesand bind synergistically to a host of integrins.

Oligopeptides were designed based on the primary and tertiary structureof human plasma fibronectin to study the structure-functionalrelationship of RGD and PHSRN regions of fibronectin in regulating thehost inflammatory response and macrophage behavior in vivo. Peptidesincluded RGD and PHSRN sequences alone or in combination. The tertiarystructure of fibronectin was utilized as a guide in the formulation ofpeptides. The distance between the PHSRN sequence and the RGD sequencewithin the natural fibronectin molecule in solution was approximatedusing the structural coordinates archived in the SwissProt Database®(sequence FINC_HUMAN P02751). Based on the measurement, a hexamer ofglycine (G₆) of approximately the same length was used to link the twobioactive sequences in both possible orientations. A terminal trimericglycine domain (G₃) was employed as a spacer in all peptides.Oligopeptides were synthesized using solid-resin methods on an automatedpeptide synthesizer (Millipore) using conventional9-fluorenylmethyloxycarbonyl chemistry without further purification andwith a final coupling efficiency of approximately ≧85% purity. Peptideswere characterized and analyzed using mass spectroscopy and reversephase HPLC coupled to photodiode array, evaporative light scatter, andUV/Vis detectors. The following oligopeptides were synthesized: G₃RGDG(SEQ. ID. NO: 3), G₃PHSRNG (SEQ. ID. NO: 4), G₃RGDG₆PHSRNG (SEQ. ID. NO:5), G₃PHSRNG₆RGDG (SEQ. ID. NO: 6), and G₃RDGG (SEQ. ID. NO: 7) as anonspecific control. Peptides were covalently grafted onto hydrogels asdescribed in Example 1 to investigate the influence of peptides on thehost response and macrophage behavior in vivo.

The gels used in this Example were random co-polymers of monomethoxypolyethyeneglycol monoacrylate (mPEGmA), acrylic acid (Ac),trimethylolpropane triacrylate (TMPTA). As noted above, these gels arehydrophilic, nonionic, low swelling, glassy, optically transparent, andcolorless. Differential scanning calorimetry analysis showed that thesematerials are completely amorphous and the mPEGmA component iscompletely phase-mixed in the cross-linked TMPTA matrix. The bioactiveoligopeptides were grafted onto mPEGmA-co-Ac-co-TMPTA hydrogels and theresulting gels mediated cell adhesion in a receptor-peptide specificmanner. The peptide surface density was found to be dependent on thenumber of amino acids per peptide. For example pentapeptides weregrafted at 66±6 pmol/cm² surface density; whereas, peptides containing30 residues were grafted at approximately one-fifth of that surfacedensity. In this Example, oligopeptides containing one bioreactiveregion (i.e., G₃RGDG, G3PHSRNG, and G₃RDGG) were grafted at about twicethe density of oligopeptides containing two bioreactive regions (i.e.,G₃RGDG₆PHSRNG and G₃PHSRNG₆RGDG).

The well-established subcutaneous cage-implant system was utilized tostudy the effect of implanted materials on the host foreign bodyreaction. Briefly, mPEGmA-co-Ac-co-TMPTA networks grafted with orwithout fibronectin-derived peptides were placed in sterile water for atleast 48 hours to remove low molecular weight leachable residualmolecules from the polymerization process and to achieve hydrationequilibrium. The polymer samples were then inserted under sterileconditions into an autoclaved cylindrical cage measured 3.5 cm long, 1cm in diameter, and constructed from medical grade stainless steel wiremesh. Cages containing various polymer samples were subcutaneouslyimplanted at the back of 3-month old female Sprague-Dawley rats. Emptycages were employed and implanted as controls. The inflammatory exudatethat collects in the cage was withdrawn at 4, 7, 10, 14, and 21 dayspost-implantation and analyzed for the quantitative evaluation ofcellular and humoral response to the test material using standard andconventional hematology techniques. Specifically, the distribution oflymphocyte, monocyte, and PMN subpopulations in the exudate wasdetermined. The presence of a high concentration of PMNs in theinflammatory exudate indicates an acute inflammatory response, whichoccurs from the onset of implantation and attenuates with time. This isfollowed by the chronic inflammatory response, which is characterized bythe presence of a high concentration of monocytes and lymphocytes in theexudate. Hence, the cage implant system allows the host inflammatoryreaction to the test sample to be observed as a function of time andmaterial property. A drop of each exudate sample was also cultured onbrain-heart infusion agar plates to check for incidence of infection. Noinfection was observed at any retrieval time for any sample. At 4, 7,14, 21, 35, and 70 days post-implantation, test polymer samples wereretrieved and the adherent cell morphology and density were quantifiedusing a video analysis system coupled to a light microscope.

A previously developed mathematical model describing the in vivokinetics of macrophage fusion on various biomaterials was employed toprovide insights into the effect of materials and peptides on foreignbody giant cell (FBGC) formation. The model was formulated based onFlory's most-probable molecular weight distribution of polymer chains.In the analysis, each adherent macrophage is analogous to a monomer andthe process of cell fusion is analogous to the polymerization process.Two initial premises are necessary: (1) the FBGC size is directlyproportional to the number of nuclei in a given FBGC; and (2) theability for each cell to fuse is constant and independent of the cellsize. The FBGC size-distribution equation (N_(x)=p^(ax-3)(1−p)) wasapplied to the measured FBGC size-distribution result of each sample ateach retrieval time. N_(x) is the cell size number-fraction of FBGCswith area x; p is the probability of cell fusion or the ratio of thenumber of cell fusion to the initial adherent macrophage density; a is aconstant relating to the number of nuclei per FBGC to the cell area(FBGC/mm²) and has been found to be constant for various clinicallyrelevant biomaterials under different mechanical stress conditions. SeeKao et al. (1994) J. Biomed. Mater. Res. 28:73-79; Kao et al. (1995) J.Biomed. Mater. Res. 29(10);1267-75; and Kao et al. (1994) J. Biomed.Mater. Res. 2:819:829. Values for p and a were obtained through acurve-fit iteration until r²>0.98. The resulting values of p for eachsample at each retrieval time were utilized to calculate two kineticparameters that characterize the process of cell fusion: the density ofadherent macrophages that participate in the FBGC formation(d₀=d_(f)/[p²(1−p)] and the rate constant of cell fusion(1/(1−p)=d₀tk+1). d₀ is the calculated density of adherent macrophagesthat participate in the FBGC formation process (macrophages/mm²), d_(f)the measured adherent macrophage density at 4 days post-implantation(macrophages ¹mm⁻²), t the implantation time (week), and k the inverserate constant of cell fusion (mm²cell⁻week⁻¹).

All experimental results are expressed in mean±standard error of themean. Each sample was independently repeated 3 times (n=3). Comparativeanalyses were performed with Statview® 4.5 using analysis of varianceand Fisher's protective t-test at 95% confidence level (p<0.05).

Total and differential leukocyte analysis was performed at severalpost-implantation periods (Table 7). No PMNs were observed at any timepoint for all samples, indicating that the presence of empty cages andnetworks grafted with or without fibronectin-derived biomimeticoligopeptides elicited a rapid acute inflammatory response that wasresolved within 4 days of implantation. For the empty cage control,total leukocyte and lymphocyte concentrations decreased rapidly between4 and 7 days post-implantation and remained steady thereafter up to 21days. Monocyte concentration remained constant from 4 to 21 dayspost-implantation. These results indicate that the presence of the emptycage elicited a rapidly decreasing chronic inflammatory response by 7days post-implantation that turned toward resolution with increasingimplantation time. The presence of mPEGmA-co-Ac-co-TMPTA gels within thecage showed a constant total leukocyte concentration from 4 to 21 daysof implantation. However, the presence of the gels increased monocyteconcentration and lowered lymphocyte concentration at days 4 and 7 whencompared with that of empty cage controls, suggesting a comparable levelof chronic inflammatory response that turned toward resolution but withan altered leukocyte sob-population distribution. When comparing thetrends between mPEGmA-co-AC-co-TMPTA networks and empty cage controls,the presence of immobilized peptides on the polymer network did notsignificantly affect the total and differential leukocyte concentrationsup to 14 days post-implantation, except that the decreased lymphocyteconcentration was not observed for G₃RGDG-grafted networks at days 4 and7 and for other peptide-grafted surfaces at day 7 of implantation.

These results indicate that the presence of polymer networks with orwithout immobilized peptides did not significantly modify the host acuteand chronic inflammatory reactions up to 14 days of implantation. By 21days of implantation, the presence of grafted G₃RGDG or G₃RDGG slightlydecreased the total and lymphocyte concentrations when compared withrespective values of “no grafted peptides” and “empty cage” controls.This trend was not observed for surfaces grafted with G₃PHSRNG₆RGDG.Conversely, the presence of grafted G₃PHSRNG or G₃RGDG₆PHSRNG slightlyincreased the total and lymphocyte concentrations when compared withrespective values of “no grafted peptides” controls (p<0.05). At 21 dayspost-implantation and thereafter, extensive fibrous encapsulation at theexterior of all implanted cages and the absence of the inflammatoryexudate inside the cage were observed for all samples, indicating theprogression of tissue healing. These data suggest that the identity ofgrafted peptides did not significantly alter the temporal variation andintensity of the host acute and chronic inflammatory reaction.

Adherent macrophage density on implanted mPEGmA-co-AC-co-TMPTA networksgrafted with or without fibronectin-derived oligopeptides was quantifiedat different retrieval times. In general, adherent macrophages on allsurfaces decreased with increasing implantation time (see Table 8).Adherent macrophage densities for all samples were comparable and werehigher than respective values of G₃RDGG or “no grafted peptide” controlsat each retrieval time up to 14 days post-implantation. Adherentmacrophage density on all samples was comparable from 21 to 70 dayspost-implantation. Adherent macrophages on all surfaces showed anextensive spread morphology with pseudopodial extension. These resultsindicate that peptides containing RGD and/or PHSRN motifs do not affectadherent macrophage density.

At each retrieval time up to 70 days post-implantation, no surfacecracking, pitting, nor other evidence of physical degradation wereobserved under polarized light microscope at 40× magnification on anypolymer sample with or without grafted peptides.

The morphology of FBGCs on all samples was that of foreign-body type,i.e., random arrangements of nuclei numbered more than three nuclei percell with widely variable, extensive cytoplasmic forms. In general, FBGCdensity increased with increasing implantation time for all samplesexcept that on surfaces grafted with G₃RGDG or G₃PHSRNG₆RGDG at whichthe adherent FBGC density remained constant with increasing implantationtime (data not shown). In addition, the average FBGC size increased withincreasing implantation time for all samples (data not shown).

These results showed that hydrogels grafted with fibronectin-derivedpeptides mediated extensive FBGC coverage that increased with increasingimplantation time. Specifically, surfaces grafted with G₃RGDG₆PHSRNGshowed the highest FBGC coverage at about 90% of the total sample areawhen compared with other sample types and controls at 70 dayspost-implantation. These in vivo findings indicate that the RGD motif,specifically in the configuration of G₃RGDG or G₃PHSRNG₆RGDG, but notG₃RGDG₆PHSRNG, modulates a rapid macrophage fusion to form FBGCs. Thisphenomenon is observed at the early stage of implantation (i.e., within4 days of implantation).

A previously developed mathematical model describing the in vivokinetics of macrophage fusion to form FBGCs on biomaterials was employedto provide insights into the effect of peptide identity on the kineticsof FBGC formation. FBGC cell size distributions on all samples weremeasured at 4, 7, 14, and 21 days post-implantation. The FBGC cellsize-distribution equation was fitted to the measured results of eachsample at each retrieval time to obtain values for p and 1/a. Values forp increased with increasing implantation time for all samples except forthat of the “no grafted peptide” controls. Thus, these results indicatethat the probability of cell fusion increased with increasingimplantation time. The calculaitons also showed that the density ofadherent macrophages that participate in the FBGC formation wassignificantly higher for mPEGmA-co-Ac-co-TMPTA gels grafted withG₃PHSRNG, and G₃PHSRNG₆RGDG than that for gels grafted with G₃RDGGnonspecific controls and gels without peptide grafting.

This Example shows that the hydrogels of the present invention can beused to support peptide, proteins, and the like, within a modified,three-dimensional hydrogel matrix. TABLE 7 Total and different leukocyteconcentration in the inflammatory exudate of mPEGmA-co-AC-co-TMPTAnetworks grafted with various fibronectin-derived oligopeptides^(a)Implantation Cell concentration (×10 cells/μl) Peptide (days) TotalLymphocyte Monocyte PMN G₃RGDG 4 127 ± 25  71 ± 22  56 ± 5^(b) 0 ± 0 7 67 ± 13  24 ± 4^(c)  43 ± 9^(b) 0 ± 0 14  74 ± 18  21 ± 4  53 ± 25 0 ±0 21  31 ± 8^(c,d,b)  27 ± 8^(c,b)  5 ± 1^(d) 0 ± 0 G₃PHSRNG 4  63 ± 32 25 ± 22^(b)  38 ± 17^(b) 0 ± 0 7  61 ± 9  25 ± 6  36 ± 3^(b) 0 ± 0 14 56 ± 19  33 ± 15  24 ± 4 0 ± 0 21  77 ± 2^(c)  69 ± 2^(c,d)  7 ± 3 0 ±0 G₃RGDG₆PHSRNG 4 129 ± 52  29 ± 10^(b)  99 ± 62^(b) 1 ± 1 7  68 ± 23 24 ± 6  44 ± 17^(b) 0 ± 0 14  57 ± 12  21 ± 9  36 ± 10 0 ± 0 21  74 ±2^(c)  67 ± 3^(c,d)  7 ± 3 0 ± 0 G₃PHSRNG₆RGDG 4 109 ± 16  53 ± 14^(b) 56 ± 5^(b) 0 ± 0 7  49 ± 11^(d)  21 ± 8  28 ± 3^(b,d) 0 ± 0 14  87 ± 23 38 ± 12  49 ± 29 0 ± 0 21  60 ± 11  55 ± 8  5 ± 3^(d) 0 ± 0 G₃RDGG 4 91 ± 11  51 ± 1^(b)  40 ± 10^(b) 0 ± 0 7  66 ± 16  30 ± 11  36 ± 6^(b)0 ± 0 14  48 ± 9^(d)  23 ± 6^(d)  25 ± 6 0 ± 0 21  35 ± 10^(c,d,b)  32 ±9^(c,b)  4 ± 2^(d) 0 ± 0 No grafted 4  94 ± 32  42 ± 27^(b)  52 ± 16^(b)0 ± 0 peptide 7  41 ± 10  11 ± 2^(b)  30 ± 6^(b) 0 ± 0 14  89 ± 21  56 ±18  33 ± 15 0 ± 0 21  63 ± 4  55 ± 4  7 ± 2^(d) 0 ± 0 Empty cage 4 135 ±22 129 ± 22  6 ± 1 0 ± 0 7  42 ± 8^(d)  38 ± 8^(d)  4 ± 1 0 ± 0 14  51 ±10^(d)  35 ± 6^(d)  15 ± 10 0 ± 0 21  82 ± 22^(d)  80 ± 25^(d)  2 ± 2 0± 0^(a)All values expressed in (mean ± s.e.m. (n = 3).^(b)Represents p < 0.05 vs. respective values of “empty cage” controls.^(c)Represents p < 0.05 vs. respective values of “no grafted peptide”controls.^(d)Represents p < 0.05 vs. respective values at day 4 of the samesample type.

TABLE 8 Adherent macrophage density on cage-implantedmPEGmA-co-AC-co-TMPTA networks grafted with various fibronectin-derivedoligopeptides^(a) Adherent macrophage density (×10 macrophages/mm²) atvarious post-implantation time (days) Peptide 4 7 14 21 35 70 G₃RGDG 138± 22^(b) 85 ± 12^(b,c) 33 ± 12^(b,c) 15 ± 3^(c) 14 ± 2^(c) 4 ± 2^(c)G₃PHSRNG 124 ± 12^(b) 57 ± 10^(b,c) 31 ± 11^(b,c) 10 ± 0^(c)  9 ± 1^(c)4 ± 2^(c) G₃EGDG₆PHSRNG 126 ± 8^(b) 58 ± 12^(b,c) 23 ± 4^(b,c) 14 ±4^(c)  6 ± 5^(c) 0 ± 0^(c) G₃PHSRNG₆RGDG 183 ± 27^(b) 69 ± 6^(b,c) 30 ±5^(b,c) 16 ± 4^(c) 12 ± 5^(c) 3 ± 1^(c) G₃RDGG  75 ± 16 36 ± 5^(c) 15 ±3^(c) 15 ± 6^(c)  9 ± 3^(c) 3 ± 2^(c) No grafted  74 ± 26 37 ± 4^(c) 14± 2^(c) 19 ± 3^(c)  6 ± 3^(c) 1 ± 1^(c) peptide^(a)All values expressed in mean ± s.e.m. (n = 3).^(b)Represents p < 0.05 vs. respective values of “no grafted peptide”controls.^(c)Represents p < 0.05 vs. respective values at day 4 of the samesample type.

Example 4 Interpenetrating Membranes Comprising Modified Hydrogels

Interpenetrating networks (IPNS) are hydrogels synthesized by reacting afirst polymer around a second material to form an intermeshingstructure. IPNs are free of cross-linkers used to create otherbiomedical hydrogels. In addition to the benefit of being free ofpotentially toxic chemicals used in conventional cross-linkingprocedures, photopolymerization has the advantages that the desiredamount of drug can be easily loaded into the matrix, and thecross-linking density, which can affect the drug release rate, can becontrolled. Furthermore, IPNs can be formed in situ and used in placesless suitable for prefabricated materials.

The focus of this Example was to investigate the swelling and drugrelease kinetics of gelatin-based IPNs of varying gelating backbonemodification, weight percent of gelatin, pH, and the molecular weight ofpolyethylene glycol diacrylate (PEGdA). Based on our results, these IPNsare quite suitable for tissue scaffolds and drug release vehicles.

Polyethyleneglycol (PEG) (Aldrich; 2, 4.6, and 8 kDa) was modified withacrylolyl chloride (Aldrich) and TEA (Aldrich) in a 1:4:6 molar ratio atroom temperature for 3 hours to produce polyethylene glycol diacrylate(PEGdA). The final PEGdA product purity was checked with the samereverse phase HPLC system as used in Example 1. The elution time of thePEGdA was approximately 13.2 minutes with a purity of approximately 100wt % PEGdA.

Monomethoxypolyethyleneglycol (mPEG) (Fluka; 2 kDa) was modified withacetic anhydride (Aldrich) and DMSO (Fisher) in a 1:80:140 molar ratioat room temperature to form an mPEG monoaldehyde (mPmA). The reactiontakes 8 to 24 hours and was monitored periodically with HPLC. The mPmAhad an elution time of approximately 11.9 minutes and a purity close to75 wt % mPmA. The compositions of PEGdA and mPEGmonoaldehyde were alsoconfirmed with ¹H-NMR.

Gelatin (G) (Sigma, Type A: from porcine skin, 300 bloom) lysyl groupswere modified with EDTAD in a 1:0.034 weight ratio for 3 hours at pH=10to form EDTAD-G (EG). Gelatin lysyl groups were also modified with mPmAand sodium cyanoborohydride (NaCNBH₃) (Aldrich) in a 1:0.66:0.186 weightratio for 24 hours at 50 to 60° C. to form mPmAG (FIG. 4). EG wasfurther modified with mPmA in a procedure similar to the mPmAGprocedure. The percent of the gelatin lysyl residues modified by EDTADand/or mPmA was determined using the trinitrobenzene sulfonic acidspectrophotometric method. The IPNs used in this study were preparedfrom the same modified gelatins.

IPNs were created using modified and unmodified gelatin, PEGdA (2, 4.6,or 8 kDa molecular weight), initiator(2,2-dimethoxy-2-phenylacetophenone, DMPA), and a long wavelength UVsource. Gelatin was dissolved in deionized water with heat (80° C.) toform a 20 wt % gelatin solution. PEGdA was dissolved in deionized water,without heat, in an aluminum foil wrapped glass vial to form a 100 wt %PEGdA solution. The gelatin solution was then added to the PEGdAsolution and the mixture was agitated thoroughly. DMPA was then added tothe gelatin/PEGdA mixture and this final mixture was again agitated andthen heated (80° C.) throughout the rest of the procedure. IPNs werecreated through injection molding. The final gelatin/PEGdA/DMPA mixturewas injected with a Pasteur pipette into a Teflon mold that was clampedbetween 2 glass slides. The mold has the approximate dimensions of 20 mmlong by 10 mm wide by 1.6 mm thick. The mold/IPN mixture was thenirradiated with UV light from the top and bottom for approximately 3minutes. During this time, the UV light initiates the cross-linking ofPEGdA, entrapping the gelatin within the PEGdA cross-links. The mold/IPNwas allowed to cool before the IPN was removed from the mold.

IPNs were named based on the weight percent of gelatin, the type ofgelatin, the weight percent of PEGdA, and the molecular weight of thePEGdA used to synthesize the IPN. For example, 4G6P2k indicates 40 wt %gelatin, 60 wt % PEGdA, 2 kDa PEGdA. The following key describes thecode used to identify IPN formulations.

Key: Each formulation is identified by a code of the formula “XYZk”,where X is the wt % gelatin, Y is the type of gelatin, Z is the wt %PEGdA, and k is the molecular weight of the PEGdA:

X wt % gelatin

-   -   4=40 wt %    -   6=60 wt %

Y=type of gelatin

-   -   G=gelatin    -   EG=EDTAD-modified gelatin    -   mPMaG=mPmA-modified gelatin    -   mPmAEG=mPmA/EDTAD-modified gelatin

Z=wt % PEGdA

-   -   4=40wt %    -   6=60 wt %

k=molecular weight PEGdA

-   -   2 k=2000 Da    -   4.6 k=4,600 Da    -   8 k=8,000 Da

The swelling/degradation kinetics of the IPNs were characterized byweighing swollen IPNs at predetermined times (up to 8 weeks). The IPNswere added to test tubes containing 5 ml deionized water withenvironmental pHs of 4.5, 7.0, and 7.4. The test tubes were then placedin water baths at 37° C. At the predetermined times, the samples wereremoved with extreme caution from the test tubes using a bent spatula,blotted dry, weighed, and then placed back in the same test tube. Thiswas done until the sample had degraded completely or until the samplehad degraded into too many pieces and they could no longer be removedfrom the test tube. The swelling weight ratio at each time point foreach IPN was calculated as: (W_(a)−W_(o)/W_(o)), where W_(s) is theweight of the swollen IPN and W_(o) is the original weight of the IPN.The maximum swelling weight ratio that occurred over 8 weeks and thetime it occurred was calculated (R_(max), T_(max)). The last attainableswelling weight ratio (due to IPN degradation) and the time it occurredwas also calculated (R_(fail), T_(fail)).

The level of host biocompatibility and inflammatory reaction of the IPNswas determined via the in vivo subcutaneous cage implant systemdescribed in the previous Examples. IPNs were placed inside cylindrical(1 cm diameter by 3.5 cm long) medical grade stainless steel wire meshcages. These cages along with empty cages, controls, were implantedsubcutaneously at the back of 3-month old female Sprague-Dawley rats.Inflammatory exudates that collected in the cages were withdrawn at 4,7, 14, and 21 days post-implantation and analyzed for the quantitativeevaluation of cellular and humoral response to the IPN samples usingstandard hematology techniques. Using these techniques the distributionsof polymorphonuclear leukocyte (PMN), lymphocyte, and monocytesubpopulations in the exudates were determined. In addition to the hostresponse, the degradation of the IPNs was determined as percent weightlost ((final IPN weight/initial IPN weight)×100).

The IPNs fabricated as described hereinabove were opaque, flexible,rubbery, and slightly tacky. The opacity increased with decreasinggelatin concentration and with increasing PEGdA molecular weight.Increasing the gelatin concentration increased the flexibility and thetackiness of the IPN. The flexibility of the IPNs also seemed toincrease with increasing PEGdA molecular weight.

The mechanical properties of the IPNs were tested using ASTM testingstandards. The IPNs for mechanical testing were made in a similarfashion as stated above, however the molds used were made ofpolydimethylsiloxane and the IPN final dimensions were 280 mm thick, 11mm gauge length, and 2 mm neck width (the dimensions required for ASTMD38-98 type IV specimens). The IPNs were subjected to tensile testingper ASTM D638-98 standards, using an Instron Model 5548 testing machine.

The preliminary mechanical tests indicated that the average Young'sModulus of the 4G6P2K IPNs was 1.26±0.14 N/nm². The ultimate tensilestress and strain were 0.39±0.10 N/nm² and 0.49±0.07 mm/mm,respectively.

Swelling/degradation studies (Table 9) showed that increasing themolecular weight of the PEGdA to 4.6 kDa and 8 kDa increased the maximumswelling ratio (R_(max)). Modifying gelatin with EDTAD and mPmA did notappear to affect R_(max). The time to R_(max) (T_(max)) increased withincreasing PEGdA molecular weight and by modifying gelatin. The swellingratio at failure (R_(fail)) decreased when the wt % of gelatin wasdecreased from 60 to 40 when PEGdA molecular weight was held constant at2 kDa. In addition, when the PEGdA molecular weight was 2 kDa and thegelatin was 60 wt %, modifying the gelatin did not improve R_(fail). Thetime to reach R_(fail) (T_(fail)) was not affected by increasing themolecular weight of PEGdA or by modifying the gelatin. Table 9 showsR_(max), T_(max), R_(fail), and T_(fail) for each composition of IPNstested at pH=7. These trends were comparable at pH of 4.5 and 7.4(results not shown). The release kinetics and bioactivity of human serumalbumin, chlorhexidine gluconate, and b-FGF (1%) from these IPNs invitro are currently being quantified. TABLE 9 Rmax, Tmax, Rfail, andTfail FOR VARIOUS IPN FORMULATIONS AT pH 7.0 Formulation R_(max) T_(max)R_(fail) T_(fail) 6G4P2K 0 0 −0.736 9 4G6P2K 0.754 1 <0.444 >13446G4P4.6K 1.733 225.667 <0.505 >1344 4G6P4.6K 2.2 4.333 <1.538 >13446G4P8K 1.646 225 0.758 451.333 4G6P8K 3.911 227.333 <1.542 >1344 6EG4P2K0.128 1 −0.214 336.33 4EG6P2K 0.712 27 <0.532 >1344 6EG4P4.6K 2.28835.333 <0.818 >1344 4EG6P4.6K 1.452 17.667 <0.773 >1344 6EG4P8K 2.639 50.794 960 4EG6P8K 3.026 1 −0.252 1097.3 6mPmAG4P2K 0.469 1.667<−0.23 >1344 4mPmAG6P2K 0.891 672 <0.827 >1344 6mPmAG4P4.6K 2.467226.667 <0.621 >1344 4mPmAG6P4.6K 2.578 616 <2.445 >1344 6mPmAG4P8K3.854 336.667 2.717 944 4mPmAG6P8K 6.075 337 <2.626 >1344 6mPmAEG4P8K2.715 2.333 <1.265 >1344 4mPmAEG6P8K 4.224 192.333 <1.472 >1344

40 wt % gelatin, 60 wt % PEGdA 2 kDa (4G6P2K) IPNs were used in apreliminary in vivo study. The presence of a high concentration of PMNin the exudates, relative to the control, indicates an acuteinflammation response, due to the onset of implantation, whichattentuates with time. Acute inflammation is followed by a highconcentration of monocytes and lymphocytes in the exudates, chronicinflammation. The study showed that there was a statistically higherinflammatory response to the IPNs after 4, 7, and 14 days ofimplantation compared to the empty cage controls. The study alsorevealed that almost 70% of the sample mass was lost after 4 days, anddecreased another 10% after 21 days.

Currently an in vivo study is underway. The study is investigating thedrug release and effect of dexamethasone from IPNs of composition 40 wt% gelatin, 60 wt % PEGdA 2 kDa, and 60 wt % PEGdA 2 kDa.

The Example illustrates that IPNs made according to the presentinvention can serve as tissue scaffolds and drug delivery vehicles.

1. A hydrogel comprising: a first polymer matrix; and a bifunctionalmodifier comprising a poly(alkylene glycol) molecule having asubstituted or unsubstituted α-terminus and a substituted orunsubstituted ω-terminus, and wherein at least one of the α- orω-termini is covalently bonded to the first polymer matrix.
 2. Thehydrogel of claim 1, further comprising a pharmacologically-active agentcovalently bonded to one of the α- or ω-termini that is not bonded tothe first polymer matrix.
 3. The hydrogel of claim 1, wherein the firstpolymer matrix is proteinaceous.
 4. The hydrogel of claim 1, wherein thefirst polymer matrix contains an amino group and wherein at least one ofthe α- or ω-termini is covalently bonded to the amino group.
 5. Thehydrogel of claim 1, wherein the first polymer matrix is selected fromthe group consisting of gelatin, calcium alginate, calcium/sodiumalginate, collagen, oxidized regenerated cellulose,carboxymethylcellulose, amino-modified cellulose, and whey protein. 6.The hydrogel of claim 1, wherein the first polymer matrix is selectedfrom the group consisting of gelatin and collagen.
 7. The hydrogel ofclaim 1, wherein the first polymer matrix is cross-linked with across-linking reagent.
 8. The hydrogel of claim 1, wherein the firstpolymer matrix is cross-linked with glutaraldehyde.
 9. The hydrogel ofclaim 1, wherein the first polymer matrix further comprises EDTADmoieties bonded to it.
 10. The hydrogel of claim 1, wherein theα-terminus and the ω-terminus of the poly(alkylene glycol) molecule aredifferent from one another.
 11. The hydrogel of claim 1, wherein theα-terminus and the (ω-terminus are substituted with a moiety selectedfrom the group consisting of halo, hydroxy, C₁-C₂₄-alkyl,C₁-C₂₄-alkenyl, C₁-C₂₄-alkynyl, C₁-C₂₄-alkoxy, C₁-C₂₄-heteroalkyl,C₁-C₂₄-heteroalkenyl, C₁-C₂₄-heteroalkynyl, cyano-C₁-C₂₄-alkyl,C₃-C₁₀-cycloalkyl, C₃-C₁₀-cycloalkenyl, C₃-C₁₀-cycloalkynyl,C₃-C₁₀-cycloheteroalkyl, C₃-C₁₀-cycloheteroalkenyl,C₃-C₁₀-cycloheteroalkynyl, acyl, acyl-C₁-C₂₄-alkyl, acyl-C₁-C₂₄-alkenyl,acyl-C₁-C₂₄-alkynyl, carboxy, C₁-C₂₄-alkylcarboxy,C₁-C₂₄-alkenylcarboxy, C₁-C₂₄-alkynylcarboxy, carboxy-C₁-C₂₄-alkyl,carboxy-C₁-C₂₄-alkenyl, carboxy-C₁-C₂₄-alkynyl, aryl, aryl-C₁-C₂₄-alkyl,aryl-C₁-C₂₄-alkenyl, aryl-C₁-C₂₄-alkynyl, heteroaryl,heteroaryl-C₁-C₂₄-alkyl, heteroaryl-C₁-C₂₄-alkenyl,heteroaryl-C₁-C₂₄-alkynyl, sulfonate, arylsulfonate, andheteroarylsulfonate.
 12. The hydrogel of claim 11, wherein the moiety onthe α-terminus is different from the moiety on the ω-terminus.
 13. Thehydrogel of claim 11, wherein the moieties on the α-terminus and theω-terminus are substituted or unsubstituted, and when substituted bear asubstituent selected from the group consisting of alkyl, aryl, acyl,halogen, hydroxy, amino, alkoxy, alkylamino, acylamino, thioamido,acyloxy, aryloxy, aryloxyalkyl, mercapto, thia, aza, oxo, saturatedcyclic hydrocarbon, unsaturated cyclic hydrocarbon, heterocycle, aryl,and heteroaryl.
 14. The hydrogel of claim 1, further comprising apharmacologically-active agent entrained within the hydrogel.
 15. Thehydrogel of claim 1, further comprising living cells entrained withinthe hydrogel.
 16. The hydrogel of claim 1, further comprising a secondpolymer matrix, wherein the second polymer matrix interpenetrates withthe first polymer matrix.
 17. The hydrogel of claim 16, wherein thesecond polymer matrix comprises a photopolymerized poly(acrylate). 18.The hydrogel of claim 16, wherein the second polymer matrix comprisesone or more monomers selected from the group consisting ofα-acrylate-ω-acrylate-poly(alkylene glycol), trimethylolpropanetriacrylate, acrylic acid, and acryloyl halide.
 19. The hydrogel ofclaim 16, further comprising a pharmacologically-active agent covalentlybonded to one of the α- or ω-termini that is not bonded to the firstpolymer matrix.
 20. The hydrogel of claim 16, further comprising apharmacologically-active agent entrained within the hydrogel.
 21. Thehydrogel of claim 16, further comprising living cells entrained withinthe hydrogel. 22-35. (canceled)
 36. A hydrogel comprising: a firstpolymer matrix containing reactive amino acid moieties; a bifunctionalmodifier comprising a compound of formula:

wherein at least one of the “A” or “Z” moieties is covalently bonded tothe reactive amino moieties of the polymer matrix; and wherein “A” and“Z” are independently selected from the group consisting of hydrogenhalo, hydroxy, C₁-C₂₄-alkyl, C₁-C₂₄-alkenyl, C₁-C₂₄-alkynyl,C₁-C₂₄-alkoxy, C₁-C₂₄-heteroalkyl. C₁-C₂₄-heteroalkenyl,C₁-C₂₄-heteroalkynyl, cyano-C₁-C₂₄-alkyl, C₃-C₁₀-alkyl,C₃-C₁₀-cycloalkenyl, C₁-C₁₀-cycloalkynyl, C₃-C₁₀-cycloheteroalkyl,C₃-C₁₀-cycloheteroalkenyl, C₃-C₁₀-cycloheteroalkynyl, acyl,acyl-C₁-C₂₄-alkyl, acyl-C₁-C₂₄-alkenyl, acyl-C₁-C₂₄-alkynyl, carboxy,C₁-C₂₄-alkylcarboxy, C₁-C₂₄-alkenylcarboxy, C₁-C₂₄-alkynylcarboxy,carboxy-C₁-C₂₄-alkyl, carboxy-C₁-C₂₄-alkenyl, carboxy-C₁-C₂₄-alkynyl,aryl, aryl-C₁-C₂₄-alkyl, aryl-C₁-C₂₄-alkenyl, aryl-C₁-C₂₄-alkynyl,heteroaryl, heteroaryl-C₁-C₂₄-alkyl, heteroaryl-C₁-C₂₄-alkenyl,heteroaryl-C₁-C₂₄-alkynyl, sulfonate, arylsulfonate, andheteroarylsulfonate; “m” is an integer of from 2 to 8; “n” is an integerequal to or greater than 100; and further comprising a second polymermatrix, wherein the second polymer matrix interpenetrates with the firstpolymer matrix.
 37. The hydrogel of claim 36, wherein the second polymermatrix comprises a photopolymerized poly(acrylate).
 38. The hydrogel ofclaim 36, wherein the second polymer matrix comprises one or moremonomers selected from the group consisting ofα-acrylate-ω-acrylate-poly(alkylene glycol), trimethylolpropanetriacrylate, and acrylic acid.
 39. The hydrogel of claim 36, furthercomprising a pharmacologically-active agent covalently bonded to one ofthe “A” or “Z” moieties that is not bonded to the first polymer matrix.40. The hydrogel of claim 36, further comprising apharmacologically-active agent entrained within the hydrogel.
 41. Thehydrogel of claim 36, further comprising living cells entrained withinthe hydrogel.
 42. A hydrogel comprising: a first polymer matrix; abifunctional modifier comprising a poly(alkylene glycol) molecule havinga substituted or unsubstituted α-terminus and a substituted orunsubstituted ω-terminus, and wherein at least one of the α- orω-termini is covalently bonded to the first polymer matrix; and a secondpolymer matrix, wherein the second polymer matrix interpenetrates withthe first polymer matrix.
 43. The hydrogel of claim 42, wherein thefirst polymer matrix is proteinaceous and the second polymer matrixcomprises a photopolymerized poly(acrylate).
 44. The hydrogel of claim42, wherein the first polymer matrix is selected from the groupconsisting of gelatin and collagen, and the second polymer matrixcomprises a photopolymerized poly(acrylate).
 45. The hydrogel of claim42, further comprising a pharmacologically-active agent covalentlybonded to one of the α- or ω-termini that is not bonded to the firstpolymer matrix.
 46. The hydrogel of claim 42, further comprising apharmacologically-active agent entrained within the hydrogel.
 47. Thehydrogel of claim 42, further comprising living cells entrained withinthe hydrogel.
 48. A method of making a hydrogel comprising: reacting afirst polymer matrix with a bifunctional modifier comprising apoly(alkylene glycol) molecule having a substituted or unsubstitutedα-terminus and a substituted or unsubstituted (a-terminus, whereby atleast one of the α- or ω-termini is covalently bonded to the polymermatrix.
 49. The method of claim 48, further comprising cross-linking thefirst polymer matrix with a cross-linking reagent.
 50. The method ofclaim 49, wherein the first polymer matrix is cross-linked withglutaraldehyde.
 51. The method of claim 48, further comprising reactingEDTAD with the first polymer matrix for a time and under conditionswherein the EDTAD binds to the polymer matrix.
 52. The method of claim48, further comprising reacting the bifunctional modifier with apharmacologically-active agent, whereby the pharmacologically-activeagent is covalently bonded to one of the the α- or ω-termini that is notbonded to the first polymer matrix.
 53. The method of claim 48, whereinthe α-terminus and the ω-terminus of the bifunctional modifier aredifferent from one another.
 54. The method of claim 48, wherein thefirst polymer matrix is selected from the group consisting of gelatin,calcium alginate, calcium/sodium alginate, collagen, oxidizedregenerated cellulose, carboxymethylcellulose, amino-modified cellulose,and whey protein.
 55. The method of claim 48, wherein the first polymermatrix is selected from the group consisting of gelatin and collagen.56. The method of claim 48, further comprising entraining apharmacologically-active agent within the hydrogel.
 57. The method ofclaim 48, further comprising contacting the first polymer matrix with aplurality of monomers and then polymerizing the monomers to yield asecond polymer matrix, wherein the second polymer matrix interpenetrateswith the first polymer matrix.
 58. The method of claim 57, wherein theplurality of monomers comprises photopolymerized poly(acrylates) and themonomers are polymerized by exposure to infrared, visible, orultraviolet radiation.
 59. The method of claim 57, wherein the pluralityof monomers comprises one or more monomers selected from the groupconsisting of α-acrylate-ω-acrylate-poly(alkylene glycol),trimethylolpropane triacrylate, acrylic acid, and acryloyl halide. 60.The method of claim 57, further comprising covalently bonding apharmacologically-active agent to one of the α- or ω-termini that is notbonded to the first polymer matrix.
 61. The method of claim 57, furthercomprising entraining a pharmacologically-active agent within thehydrogel.
 62. The method of claim 57, further comprising entrainingliving cells within the hydrogel.
 63. A method of administeringpharmacologically-active agents or cells to a patient in need thereof,the method comprising entraining pharmacologically-active agents orcells within a hydrogel as recited in claim 1, and then administeringthe hydrogel to a patient in need of the pharmacologically-active agentor cells.
 64. A method of administering pharmacologically-active agentsor cells to a patient in need thereof, the method comprising entrainingpharmacologically-active agents or cells within a hydrogel as recited inclaim 22, and then administering the hydrogel to a patient in need ofthe pharmacologically-active agent or cells.
 65. A method ofadministering pharmacologically-active agents or cells to a patient inneed thereof, the method comprising entraining pharmacologically-activeagents or cells within a hydrogel as recited in claim 42, and thenadministering the hydrogel to a patient in need of thepharmacologically-active agent or cells.